In Vitro sorting method

ABSTRACT

The invention describes a method for isolating one or more genetic elements encoding a gene product having a desired activity, comprising of the steps of: compartmentalizing genetic elements into microcapsules; expressing the genetic elements to produce their respective gene products within the microcapsules; sorting the genetic elements which produce the gene product having a desired activity. The invention enables the in vitro evolution of nucleic acids by repeated mutagenesis and iterative applications of the method of the invention.

This application is a divisional of U.S. patent application Ser. No.10/263,984, filed Oct. 3, 2002, now issued as U.S. Pat. No. 7,138,233,which claims the priority of U.S. application Ser. No. 09/464,122, filedDec. 16, 1999, now issued as U.S. Pat. No. 6,489,103, and ofinternational application PCT/GB98/01889, filed Jun. 29, 1998 andpublished in English, which application claimed the priority of GreatBritain Application Nos. GB9714300.2, filed Jul. 7, 1997 andGB9806393.6, filed Mar. 25, 1998.

The present invention relates to methods for use in in vitro evolutionof molecular libraries. In particular, the present invention relates tomethods of selecting nucleic acids encoding gene products in which thenucleic acid and the activity of the encoded gene product are linked bycompartmentation.

Evolution requires the generation of genetic diversity (diversity innucleic acid) followed by the selection of those nucleic acids whichresult in beneficial characteristics. Because the nucleic acid and theactivity of the encoded gene product of an organism are physicallylinked (the nucleic acids being confined within the cells which theyencode) multiple rounds of mutation and selection can result in theprogressive survival of organisms with increasing fitness. Systems forrapid evolution of nucleic acids or proteins in vitro must mimic thisprocess at the molecular level in that the nucleic acid and the activityof the encoded gene product must be linked and the activity of the geneproduct must be selectable.

Recent advances in molecular biology have allowed some molecules to beco-selected according to their properties along with the nucleic acidsthat encode them. The selected nucleic acids can subsequently be clonedfor further analysis or use, or subjected to additional rounds ofmutation and selection.

Common to these methods is the establishment of large libraries ofnucleic acids. Molecules having the desired characteristics (activity)can be isolated through selection regimes that select for the desiredactivity of the encoded gene product, such as a desired biochemical orbiological activity, for example binding activity.

Phage display technology has been highly successful as providing avehicle that allows for the selection of a displayed protein byproviding the essential link between nucleic acid and the activity ofthe encoded gene product (Smith, 1985; Bass et al. , 1990; McCafferty etal., 1990; for review see Clackson and Wells, 1994). Filamentous phageparticles act as genetic display packages with proteins on the outsideand the genetic elements which encode them on the inside. The tightlinkage between nucleic acid and the activity of the encoded geneproduct is a result of the assembly of the phage within bacteria. Asindividual bacteria are rarely multiply infected, in most cases all thephage produced from an individual bacterium will carry the same geneticelement and display the same protein.

However, phage display relies upon the creation of nucleic acidlibraries in vivo in bacteria. Thus, the practical limitation on librarysize allowed by phage display technology is of the order of 10⁷ to 10¹¹,even taking advantage of λ phage vectors with excisable filamentousphage replicons. The technique has mainly been applied to selection ofmolecules with binding activity. A small number of proteins withcatalytic activity have also been isolated using this technique,however, in no case was selection directly for the desired catalyticactivity, but either for binding to a transition-state analogue(Widersten and Mannervik, 1995) or reaction with a suicide inhibitor(Soumillion et al., 1994; Janda et al., 1997).

Specific peptide ligands have been selected for binding to receptors byaffinity selection using large libraries of peptides linked to the Cterminus of the lac repressor Lacl (Cull et al., 1992). When expressedin E. coli the repressor protein physically links the ligand to theencoding plasmid by binding to a lac operator sequence on the plasmid.

An entirely in vitro polysome display system has also been reported(Mattheakis et al., 1994) in which nascent peptides are physicallyattached via the ribosome to the RNA which encodes them.

However, the scope of the above systems is limited to the selection ofproteins and furthermore does not allow direct selection for activitiesother than binding, for example catalytic or regulatory activity.

In vitro RNA selection and evolution (Ellington and Szostak, 1990),sometimes referred to as SELEX (systematic evolution of ligands byexponential enrichment) (Tuerk and Gold, 1990) allows for selection forboth binding and chemical activity, but only for nucleic acids. Whenselection is for binding, a pool of nucleic acids is incubated withimmobilised substrate. Non-binders are washed away, then the binders arereleased, amplified and the whole process is repeated in iterative stepsto enrich for better binding sequences. This method can also be adaptedto allow isolation of catalytic RNA and DNA (Green and Szostak, 1992;for reviews see Chapman and Szostak, 1994. Joyce, 1994; Gold et al.,1995; Moore, 1995).

However, selection for “catalytic” or binding activity using SELEX isonly possible because the same molecule performs the dual role ofcarrying the genetic information and being the catalyst or bindingmolecule (aptamer). When selection is for “auto-catalysis” the samemolecule must also perform the third role of being a substrate. Sincethe genetic element must play the role of both the substrate and thecatalyst, selection is only possible for single turnover events. Becausethe “catalyst” is in this process itself modified, it is by definitionnot a true catalyst. Additionally, proteins may not be selected usingthe SELEX procedure. The range of catalysts, substrates and reactionswhich can be selected is therefore severely limited.

Those of the above methods that allow for iterative rounds of mutationand selection are mimicking in vitro mechanisms usually ascribed to theprocess of evolution: iterative variation, progressive selection for adesired the activity and replication. However, none of the methods sofar developed have provided molecules of comparable diversity andfunctional efficacy to those that are found naturally. Additionally,there are no man-made “evolution” systems which can evolve both nucleicacids and proteins to effect the full range of biochemical andbiological activities (for example, binding, catalytic and regulatoryactivities) and that can combine several processes leading to a desiredproduct or activity.

There is thus a great need for an in vitro system that overcomes thelimitations discussed above.

BRIEF DESCRIPTION OF THE INVENTION

According to a first aspect of the present invention, there is provideda method for isolating one or more genetic elements encoding a geneproduct having a desired activity, comprising the steps of:

-   -   (a) compartmentalising genetic elements into microcapsules;    -   (b) expressing the genetic elements to produce their respective        gene products within the microcapsules;    -   (c) sorting the genetic elements which produce the gene        product(s) having the desired activity.

The microcapsules according to the present invention compartmentalisegenetic elements and gene products such that they remain physicallylinked together. Surprisingly, nucleic acid expression remains possiblewithin the artificial microcapsules allowing for isolation of nucleicacid on the basis if the activity of the gene product which it encodes.

As used herein, a genetic element is a molecule or molecular constructcomprising a nucleic acid. The genetic elements of the present inventionmay comprise any nucleic acid (for example, DNA, RNA or any analogue,natural or artificial, thereof). The nucleic acid component of thegenetic element may moreover be linked, covalently or non-covalently, toone or more molecules or structures, including proteins, chemicalentities and groups, solid-phase supports such as magnetic beads, andthe like. In the method of the invention, these structures or moleculescan be designed to assist in the sorting and/or isolation of the geneticelement encoding a gene product with the desired activity.

Expression, as used herein, is used in its broadest meaning, to signifythat a nucleic acid contained in the genetic element is converted intoits gene product. Thus, where the nucleic acid is DNA, expression refersto the transcription of the DNA into RNA; where this RNA codes forprotein, expression may also refer to the translation of the RNA intoprotein. Where the nucleic acid is RNA, expression may refer to thereplication of this RNA into further RNA copies, the reversetranscription of the RNA into DNA and optionally the transcription ofthis DNA into further RNA molecule(s), as well as optionally thetranslation of any of the RNA species produced into protein. Preferably,therefore, expression is performed by one or more processes selectedfrom the group consisting of transcription, reverse transcription,replication and translation.

Expression of the genetic element may thus be directed into either DNA,RNA or protein, or a nucleic acid or protein containing unnatural basesor amino acids (the gene product) within the microcapsule of theinvention, so that the gene product is confined within the samemicrocapsule as the genetic element.

The genetic element and the gene product thereby encoded are linked byconfining each genetic element and the respective gene product encodedby the genetic element within the same microcapsule. In this way thegene product in one microcapsule cannot cause a change in any othermicrocapsules.

The term “microcapsule” is used herein in accordance with the meaningnormally assigned thereto in the art and further described hereinbelow.In essence, however, a microcapsule is an artificial compartment whosedelimiting borders restrict the exchange of the components of themolecular mechanisms described herein which allow the sorting of geneticelements according to the function of the gene products which theyencode.

Preferably, the microcapsules used in the method of the presentinvention will be capable of being produced in very large numbers, andthereby to compartmentalise a library of genetic elements which encodesa repertoire of gene products.

According to a preferred embodiment of the first aspect of the presentinvention, the sorting of genetic elements may be performed in one ofessentially four techniques.

(I) In a first embodiment, the microcapsules are sorted according to anactivity of the gene product or derivative thereof which makes themicrocapsule detectable as a whole. Accordingly, the invention providesa method according to the first aspect of the invention wherein a geneproduct with the desired activity induces a change in the microcapsule,or a modification of one or more molecules within the microcapsule,which enables the microcapsule containing the gene product and thegenetic element encoding it to be sorted. In this embodiment, therefore,the microcapsules are physically sorted from each other according to theactivity of the gene product(s) expressed from the genetic element(s)contained therein, which makes it possible selectively to enrich formicrocapsules containing gene products of the desired activity.

(II) In a second embodiment, the genetic elements are sorted followingpooling of the microcapsules into one or more common compartments. Inthis embodiment, a gene product having the desired activity modifies thegenetic element which encoded it (and which resides in the samemicrocapsule) in such a way as to make it selectable in a subsequentstep. The reactions are stopped and the microcapsules are then broken sothat all the contents of the individual microcapsules are pooled.Selection for the modified genetic elements enables enrichment of thegenetic elements encoding the gene product(s) having the desiredactivity. Accordingly, the invention provides a method according to thefirst aspect of the invention, wherein in step (b) the gene producthaving the desired activity modifies the genetic element encoding it toenable the isolation of the genetic element. It is to be understood, ofcourse, that modification may be direct, in that it is caused by thedirect action of the gene product on the genetic element, or indirect,in which a series of reactions, one or more of which involve the geneproduct having the desired activity, leads to modification of thegenetic element.

(III) In a third embodiment, the genetic elements are sorted followingpooling of the microcapsules into one or more common compartments. Inthis embodiment, a gene with a desired activity induces a change in themicrocapsule containing the gene product and the genetic elementencoding it. This change, when detected, triggers the modification ofthe gene within the compartment. The reactions are stopped and themicrocapsules are then broken so that all the contents of the individualmicrocapsules are pooled. Selection for the modified genetic elementsenables enrichment of the genetic elements encoding the gene product(s)having the desired activity. Accordingly the invention provides a methodaccording to the first aspect of the invention, where in step (b) thegene product having the desired activity induces a change in thecompartment which is detected and triggers the modification of thegenetic element within the compartment so as to allow its isolation. Itis to be understood that the detected change in the compartment may becaused by the direct action of the gene product, or indirect action, inwhich a series of reactions, one or more of which involve the geneproduct having the desired activity leads to the detected change.

(IV) In a fourth embodiment, the genetic elements may be sorted by amulti-step procedure, which involves at least two steps, for example, inorder to allow the exposure of the genetic elements to conditions whichpermit at least two separate reactions to occur. As will be apparent toa persons skilled in the art, the first microencapsulation step of theinvention must result in conditions which permit the expression of thegenetic elements—be it transcription, transcription and/or translation,replication or the like. Under these conditions, it may not be possibleto select for a particular gene product activity, for example becausethe gene product may not be active under these conditions, or becausethe expression system contains an interfering activity. The inventiontherefore provides a method according to the first aspect of the presentinvention, wherein step (b) comprises expressing the genetic elements toproduce their respective gene products within the microcapsules, linkingthe gene products to the genetic elements encoding them and isolatingthe complexes thereby formed. This allows for the genetic elements andtheir associated gene products to be isolated from the capsules beforesorting according to gene product activity takes place. In a preferredembodiment, the complexes are subjected to a furthercompartmentalisation step prior to isolating the genetic elementsencoding a gene product having the desired activity. This furthercompartmentalisation step, which advantageously takes place inmicrocapsules, permits the performance of further reactions, underdifferent conditions, in an environment where the genetic elements andtheir respective gene products are physically linked. Eventual sortingof genetic elements may be performed according to embodiment (I), (II)or (III) above.

The “secondary encapsulation” may also be performed with geneticelements linked to gene products by other means, such as by phagedisplay, polysome display, RNA-peptide fusion or lac repressor peptidefusion.

The selected genetic element(s) may also be subjected to subsequent,possibly more stringent rounds of sorting in iteratively repeated steps,reapplying the method of the invention either in its entirety or inselected steps only. By tailoring the conditions appropriately, geneticelements encoding gene products having a better optimised activity maybe isolated after each round of selection.

Additionally, the genetic elements isolated after a first round ofsorting may be subjected to mutagenesis before repeating the sorting byiterative repetition of the steps of the method of the invention as setout above. After each round of mutagenesis, some genetic elements willhave been modified in such a way that the activity of the gene productsis enhanced.

Moreover, the selected genetic elements can be cloned into an expressionvector to allow further characterisation of the genetic elements andtheir products.

In a second aspect, the invention provides a product when selectedaccording to the first aspect of the invention. As used in this context,a “product” may refer to a gene product, selectable according to theinvention, or the genetic element (or genetic information comprisedtherein).

In a third aspect, the invention provides a method for preparing a geneproduct, comprising the steps of:

-   -   (a) preparing a genetic element encoding the gene product;    -   (b) compartmentalising genetic elements into microcapsules;    -   (c) expressing the genetic elements to produce their respective        gene products within the microcapsules;    -   (d) sorting the genetic elements which produce the gene        product(s) having the desired activity; and    -   (e) expressing the gene product having the desired activity.

In accordance with the third aspect, step (a) preferably comprisespreparing a repertoire of genetic elements, wherein each genetic elementencodes a potentially differing gene product. Repertoires may begenerated by conventional techniques, such as those employed for thegeneration of libraries intended for selection by methods such as phagedisplay. Gene products having the desired activity may be selected fromthe repertoire, according to the present invention.

In a fourth aspect, the invention provides a method for screening acompound or compounds capable of modulation the activity of a geneproduct, comprising the steps of:

-   -   (a) preparing a repertoire of genetic element encoding gene        product;    -   (b) compartmentalising genetic elements into microcapsules;    -   (c) expressing the genetic elements to produce their respective        gene products within the microcapsules;    -   (d) sorting the genetic elements which produce the gene        product(s) having the desired activity; and    -   (e) contacting a gene product having the desired activity with        the compound or compounds and monitoring the modulation of an        activity of the gene product by the compound or compounds.

Advantageously, the method further comprises the step of:

-   -   (f) identifying the compound or compounds capable of modulating        the activity of the gene product and synthesising said compound        or compounds.

This selection system can be configured to select for RNA, DNA orprotein molecules with catalytic, regulatory or binding activity.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1

Gene Selection by Compartmentalisation.

A. Schematic representation of the selection procedure. In Step 1, an invitro transcription/translation reaction mixture containing a library ofgenetic elements linked to a substrate for the reaction being selectedis dispersed to form a water-in-oil emulsion with typically one geneticelement per aqueous compartment. The genetic elements are transcribedand translated within their compartments (Step 2). Subsequently (Step3), proteins (or RNAs) with enzymatic activities convert the substrateinto a product that remains liked to the genetic element.Compartnentalisation prevents the modification of genetic elements inother compartments. Next (Step 4), the emulsion is broken, all reactionsare stopped and the aqueous compartments combined. Genetic elementswhich are linked to the product are selectively enriched, thenamplified, and either characterised (Step 5), or linked to the substrateand compartnentalised for further rounds of selection (Step 6).

B. Selection for target-specific DNA methylation by HaeIII methylase.The substrate is a segment of DNA containing HaeIIIrestriction/modification (R/M) sites. Genetic elements are isolated bybinding to streptavidin—coated magnetic beads and treated with thecognate restriction enzyme HaeIII. Only nucleic acids with methylatedR/M sites are resistant to cleavage and subsequently amplified by PCR.

FIG. 2

A. Droplet size distribution and activities of DHFR and HaeIII methylasein emulsions: size distribution of the aqueous compartments in anemulsion determined by laser diffraction. In vitrotranscription/translation reaction mixtures containing DNA and sodiumdeoxycholate are emulsified by stirring, or by stirring followed byhomogenisation at 8 k, 9.5 k or 13.5 k rpm. The size distribution of theaqueous particles is shown by percentage of the total aqueous volume. BThe activity of DHFR formed in situ by transcription and translation ofits gene (FIG. 1B) in aqueous compartments of an emulsion. Theconcentration of the folA gene used (2.5 nM) gives an average of onegene per droplet in the finest emulsions (homogenised at 13.5 k rpm).The mean diameter calculated from the size distribution data (in FIG. 2)is presented as a function of the speed of homogenisation (0 k rpmrefers to the emulsion prepared by stirring with no furtherhomogenisation). Activity is presented as percentage of the activityobserved in the non-emulsified in vitro reaction mixture under the sameconditions. The activity of HaeIII methylase formed in situ bytranscription and translation of its gene (FIG. 1B) in aqueouscompartments of an emulsion. The concentration of the M.HaeIII gene used(2.5 nM) gives an average of one gene per droplet in the finestemulsions (homogenised at 13.5 k rpm). The mean diameter calculated fromthe size distribution data (in FIG. 2A) is presented as a function ofthe speed of homogenisation; (0 k rpm refers to the emulsion prepared bystirring with no further homogenisation). Activity is presented aspercentage of the activity observed in the non-emulsified in vitroreaction mixture under the same conditions.

FIG. 3

Selections for HaeIII DNA Methylase.

A Selecting M.HaeIII genes from a 1000-fold excess of folA genes.Reactions were set up with 0.2 nM of DIG-folA-3s-Biotin DNA(corresponding to an average of one gene per compartment), spiked with0. 2 pM of DIG-M.HaeIII-3s-Biotin. Reaction mixtures were eitheremulsified by stirring or left in solution. The DNA from these reactionswas captured, digested with HaeIII (or with HhaI) and amplified by PCR.This DNA was further amplified by nested PCR with primers LMB2-Nest andLMB3-Nest and five microlitres of each nested PCR was electrophoresed ona 1.5% agarose gel containing ethidium bromide. Markers, ΦX174-HaeIIIdigest; minus T7, no T7 RNA polymerase; minus NadCh, no sodiumdeoxycholate. B Two-round selections. Reactions containing a 1:10⁴ to1:10⁷ molar ratio of DIG-M.HaeIII-3s-Biotin: DIG-folA-3s- Biotin (at 500pM) are emulsified by stirring. The DNA from these reactions is digestedwith HaeIII and amplified by PCR with primers LMB2-Biotin (SEQ. ID. No.9) and LMB3-DIG (SEQ. ID. NO. 10). The amplified DNA from the firstround selection of 1:10⁴ and 1:10₅ ratios (at 20 pM) and the 1:10₆ and1:10₇ ratios (at 500 pM) is put into a second round of selection. ThisDNA was further amplified by nested PCR with primers LMB2-Nest andLMB3-Nest and five microlitres of nested PCR from each round ofselection are analysed by gel electrophoresis as above (upper panel).The same DNA was translated in vitro and the resulting methylaseactivity was measured. Results are presented as the percentage ofsubstrate DNA methylated (lower panel).

DETAILED DESCRIPTION OF THE INVENTION

(A) General Description

The microcapsules of the present invention require appropriate physicalproperties to allow the working of the invention.

First, to ensure that the genetic elements and gene products may notdiffuse between microcapsules, the contents of each microcapsule must beisolated from the contents of the surrounding microcapsules, so thatthere is no or little exchange of the genetic elements and gene productsbetween the microcapsules over the timescale of the experiment.

Second, the method of the present invention requires that there are onlya limited number of genetic elements per microcapsule. This ensures thatthe gene product of an individual genetic element will be isolated fromother genetic elements. Thus, coupling between genetic element and geneproduct will be highly specific. The enrichment factor is greatest withon average one or fewer genetic elements per microcapsule, the linkagebetween nucleic acid and the activity of the encoded gene product beingas tight as is possible, since the gene product of an individual geneticelement will be isolated from the products of all other geneticelements. However, even if the theoretically optimal situation of, onaverage, a single genetic element or less per microcapsule is not used,a ratio of 5, 10, 50, 100 or 1000 or more genetic elements permicrocapsule may prove beneficial in sorting a large library. Subsequentrounds of sorting, including renewed encapsulation with differinggenetic element distribution, will permit more stringent sorting of thegenetic elements. Preferably, there is a single genetic element, orfewer, per microcapsule.

Third, the formation and the composition of the microcapsules must notabolish the function of the machinery the expression of the geneticelements and the activity of the gene products.

Consequently, any microencapsulation system used must fulfil these threerequirements. The appropriate system(s) may vary depending on theprecise nature of the requirements in each application of the invention,as will be apparent to the skilled person.

A wide variety of microencapsulation procedures are available (seeBenita, 1996) and may be used to create the microcapsules used inaccordance with the present invention. Indeed, more than 200microencapsulation methods have been identified in the literature(Finch, 1993).

These include membrane enveloped aqueous vesicles such as lipid vesicles(liposomes) (New, 1990) and non-ionic surfactant vesicles (van Hal etal., 1996). These are closed-membranous capsules of single or multiplebilayers of non-covalently assembled molecules, with each bilayerseparated from its neighbour by an aqueous compartment. In the case ofliposomes the membrane is composed of lipid molecules; these are usuallyphospholipids but sterols such as cholesterol may also be incorporatedinto the membranes (New, 1990). A variety of enzyme-catalysedbiochemical reactions, including RNA and DNA polymerisation, can beperformed within liposomes (Chakrabarti et al., 1994; Oberholzer et al.,1995a; Oberholzer et al., 1995b; Walde et al., 1994; Wick & Luisi,1996).

With a membrane-enveloped vesicle system much of the aqueous phase isoutside the vesicles and is therefore non-compartmentalised. Thiscontinuous, aqueous phase should be removed or the biological systems init inhibited or destroyed (for example, by digestion of nucleic acidswith DNase or RNase) in order that the reactions are limited to themicrocapsules (Luisi et al., 1987).

Enzyme-catalysed biochemical reactions have also been demonstrated inmicrocapsules generated by a variety of other methods. Many enzymes areactive in reverse micellar solutions (Bru & Walde, 1991; Bru & Walde,1993; Creagh et al., 1993; Haber et al., 1993; Kumar et al., 1989; Luisi& B., 1987; Mao & Walde, 1991; Mao et al., 1992; Perez et al., 1992;Walde et al., 1994; Walde et al., 1993; Walde et al., 1988) such as theAOT-isooctane-water system (Menger & Yamada, 1979).

Microcapsules can also be generated by interfacial polymerisation andinterfacial complexation (Whateley, 1996). Microcapsules of this sortcan have rigid, nonpermeable membranes, or semipermeable membranes.Semipermeable microcapsules bordered by cellulose nitrate membranes,polyamide membranes and lipid-polyamide membranes can all supportbiochemical reactions, including multienzyme systems (Chang, 1987;Chang, 1992; Lim, 1984). Alginate/polylysine microcapsules (Lim & Sun,1980), which can be formed under very mild conditions, have also provento be very biocompatible, providing, for example, an effective method ofencapsulating living cells and tissues (Chang, 1992; Sun et al., 1992).

Non-membranous microencapsulation systems based on phase partitioning ofan aqueous environment in a colloidal system, such as an emulsion, mayalso be used.

Preferably, the microcapsules of the present invention are formed fromemulsions; heterogeneous systems of two immiscible liquid phases withone of the phases dispersed in the other as droplets of microscopic orcolloidal size (Becher, 1957; Sherman, 1968; Lissant, 1974; Lissant,1984).

Emulsions may be produced from any suitable combination of immiscibleliquids. Preferably the emulsion of the present invention has water(containing the biochemical components) as the phase present in the formof finely divided droplets (the disperse, internal or discontinuousphase) and a hydrophobic, immiscible liquid (an ‘oil’) as the matrix inwhich these droplets are suspended (the nondisperse, continuous orexternal phase). Such emulsions are termed ‘water-in-oil’ (W/O). Thishas the advantage that the entire aqueous phase containing thebiochemical components is compartmentalised in discreet droplets (theinternal phase). The external phase, being a hydrophobic oil, generallycontains none of the biochemical components and hence is inert.

The emulsion may be stabilised by addition of one or more surface-activeagents (surfactants). These surfactants are termed emulsifying agentsand act at the water/oil interface to prevent (or at least delay)separation of the phases. Many oils and many emulsiflers can be used forthe generation of water-in-oil emulsions; a recent compilation listedover 16,000 surfactants, many of which are used as emulsifying agents(Ash and Ash, 1993). Suitable oils include light white mineral oil andnon-ionic surfactants (Schick, 1966) such as sorbitan monooleate (SPAN®80; ICI) and polyoxyethylenesorbitan monooleate (TWEEN® 80; ICI).

The use of anionic surfactants may also be beneficial. Suitablesurfactants include sodium cholate and sodium taurocholate. Particularlypreferred is sodium deoxycholate, preferably at a concentration of 0.5%w/v, or below. Inclusion of such surfactants can in some cases increasethe expression of the genetic elements and/or the activity of the geneproducts. Addition of some anionic surfactants to a non-emulsifiedreaction mixture completely abolishes translation. Duringemulsification, however, the surfactant is transferred from the aqueousphase into the interface and activity is restored. Addition of ananionic surfactant to the mixtures to be emulsified ensures thatreactions proceed only after compartmentalisation.

Creation of an emulsion generally requires the application of mechanicalenergy to force the phases together. There are a variety of ways ofdoing this which utilise a variety of mechanical devices, includingstirrers (such as magnetic stir-bars, propeller and turbine stirrers,paddle devices and whisks), homogenisers (including rotor-statorhomogenisers, high-pressure valve homogenisers and jet homogenisers),colloid mills, ultrasound and ‘membrane emulsification’ devices (Becher,1957; Dickinson, 1994).

Aqueous microcapsules formed in water-in-oil emulsions are generallystable with little if any exchange of genetic elements or gene productsbetween microcapsules. Additionally, we have demonstrated that severalbiochemical reactions proceed in emulsion microcapsules. Moreover,complicated biochemical processes, notably gene transcription andtranslation are. also active in emulsion microcapsules. The technologyexists to create emulsions with volumes all the way up to industrialscales of thousands of litres (Becher, 1957; Sherman, 1968; Lissant,1974; Lissant, 1984).

The preferred microcapsule size will vary depending upon the preciserequirements of any individual selection process that is to be performedaccording to the present invention. In all cases, there will be anoptimal balance between gene library size, the required enrichment andthe required concentration of components in the individual microcapsulesto achieve efficient expression and reactivity of the gene products.

The processes of expression must occur within each individualmicrocapsule provided by the present invention. Both in vitrotranscription and coupled transcription-translation become lessefficient at sub-nanomolar DNA concentrations. Because of therequirement for only a limited number of DNA molecules to be present ineach microcapsule, this therefore sets a practical upper limit on thepossible microcapsule size. Preferably, the mean volume of themicrocapsules is less that 5.2×10⁻¹⁶ m³, (corresponding to a sphericalmicrocapsule of diameter less than 10 μm, more preferably less than6.5×10⁻¹⁷ m³ (5 μm), more preferably about 4.2×10⁻¹⁸ m³ (2 μm) andideally about 9×10⁻¹⁸ m³ (2.6 μm).

The effective DNA or RNA concentration in the microcapsules may beartificially increased by various methods that will be well-known tothose versed in the art. These include, for example, the addition ofvolume excluding chemicals such as polyethylene glycols (PEG) and avariety of gene amplification techniques, including transcription usingRNA polymerases including those from bacteria such as E. coli (Roberts,1969; Blattner and Dahlberg, 1972; Roberts et al., 1975; Rosenberg etal. , 1975), eukaryotes e. g. (Weil et al. , 1979; Manley et al., 1983)and bacteriophage such as T7, T3 and SP6 (Melton et al., 1984); thepolymerase chain reaction (PCR) (Saiki et al., 1988); Qβ replicaseamplification (Miele et al., 1983; Cahill et al., 1991; Chetverin andSpirin, 1995; Katanaev et al., 1995); the ligase chain reaction (LCR)(Landegren et al., 1988; Barany, 1991); and self-sustained sequencereplication system (Fahy et al., 1991) and strand displacementamplification (Walker et al., 1992). Even gene amplification techniquesrequiring thermal cycling such as PCR and LCR could be used if theemulsions and the in vitro transcription or coupledtranscription-translation systems are thermostable (for example, thecoupled transcription-translation systems could be made from athermostable organism such as Thermus aquaticus).

Increasing the effective local nucleic acid concentration enables largermicrocapsules to be used effectively. This allows a preferred practicalupper limit to the microcapsule volume of about 5.2×10⁻¹⁶ m³(corresponding to a sphere of diameter 10 μm).

The microcapsule size must be sufficiently large to accommodate all ofthe required components of the biochemical reactions that are needed tooccur within the microcapsule. For example, in vitro, both transcriptionreactions and coupled transcription-translation reactions require atotal nucleoside triphosphate concentration of about 2 mM.

For example, in order to transcribe a gene to a single short RNAmolecule of 500 bases in length, this would require a minimum of 500molecules of nucleoside triphosphate per microcapsule (8.33×10⁻²²moles). In order to constitute a 2 mM solution, this number of moleculesmust be contained within a microcapsule of volume 4.17×10⁻¹⁹ litres(4.17×10⁻²² m³ which if spherical would have a diameter of 93 nm.

Furthermore, particularly in the case of reactions involvingtranslation, it is to be noted that the ribosomes necessary for thetranslation to occur are themselves approximately 20 nm in diameter.Hence, the preferred lower limit for microcapsules is a diameter ofapproximately 0.1 μm (100 nm).

Therefore, the microcapsule volume is preferably of the order of between5.2×10⁻²² m³ and 5.2×10⁻¹⁶ m³ corresponding to a sphere of diameterbetween 0.1 μm and 10 μm, more preferably of between about 5.2×10⁻¹⁹ m³and 6.5×10⁻¹⁷ m³ (1 μm and 5 μm). Sphere diameters of about 2.6 μm aremost advantageous.

It is no coincidence that the preferred dimensions of the compartments(droplets of 2.6 μm mean diameter) closely resemble those of bacteria,for example, Escherichia are 1.1-1.5×2.0-6.0 μm rods and Azotobacter are1.5-2.0 μm diameter ovoid cells. In its simplest form, Darwinianevolution is based on a ‘one genotype one phenotype’ mechanism. Theconcentration of a single compartmentalised gene, or genome, drops from0.4 nM in a compartment of 2 μm diameter, to 25 pM in a compartment of 5μm diameter. The prokaryotic transcription/translation machinery hasevolved to operate in compartments of˜1-2 μm diameter, where singlegenes are at approximately nanomolar concentrations. A single gene, in acompartment of 2.6 μm diameter is at a concentration of 0.2 nM. Thisgene concentration is high enough for efficient translation.Compartmentalisation in such a volume also ensures that even if only asingle molecule of the gene product is formed it is present at about 0.2nM, which is important if the gene product is to have a modifyingactivity of the genetic element itself. The volume of the microcapsuleshould thus be selected bearing in mind not only the requirements fortranscription and translation of the genetic element, but also themodifying activity required of the gene product in the method of theinvention.

The size of emulsion microcapsules may be varied simply by tailoring theemulsion conditions used to form the emulsion according to requirementsof the selection system. The larger the microcapsule size, the larger isthe volume that will be required to encapsulate a given genetic elementlibrary, since the ultimately limiting factor will be the size of themicrocapsule and thus the number of microcapsules possible per unitvolume.

The size of the microcapsules is selected not only having regard to therequirements of the transcription/translation system, but also those ofthe selection system employed for the genetic element. Thus, thecomponents of the selection system, such as a chemical modificationsystem, may require reaction volumes and/or reagent concentrations whichare not optimal for transcription/translation. As set forth herein, suchrequirements may be accommodated by a secondary re-encapsulation step;moreover, they may be accommodated by selecting the microcapsule size inorder to maximise transcription/translation and selection as a whole.Empirical determination of optimal microcapsule volume and reagentconcentration, for example as set forth herein, is preferred.

A “genetic element” in accordance with the present invention is asdescribed above. Preferably, a genetic element is a molecule orconstruct selected from the group consisting of a DNA molecule, an RNAmolecule, a partially or wholly artificial nucleic acid moleculeconsisting of exclusively synthetic or a mixture of naturally-occurringand synthetic bases, any one of the foregoing linked to a polypeptide,and any one of the foregoing linked to any other molecular group orconstruct. Advantageously, the other molecular group or construct may beselected from the group consisting of nucleic acids, polymericsubstances, particularly beads, for example polystyrene beads, magneticsubstances such as magnetic beads, labels, such as fluorophores orisotopic labels, chemical reagents, binding agents such as macrocyclesand the like.

The nucleic acid portion of the genetic element may comprise suitableregulatory sequences, such as those required for efficient expression ofthe gene product, for example promoters, enhancers, translationalinitiation sequences, polyadenylation sequences, splice sites and thelike.

As will be apparent from the following, in many cases the polypeptide orother molecular group or construct is a ligand or a substrate whichdirectly or indirectly binds to or reacts with the gene product in orderto tag the genetic element. This allows the sorting of the geneticelement on the basis of the activity of the gene product.

The ligand or substrate can be connected to the nucleic acid by avariety of means that will be apparent to those skilled in the art (see,for example, Hermanson, 1996). Any tag will suffice that allows for thesubsequent selection of the genetic element. Sorting can be by anymethod which allows the preferential separation, amplification orsurvival of the tagged genetic element. Examples include selection bybinding (including techniques based on magnetic separation, for exampleusing Dynabeads™), and by resistance to degradation (for example bynucleases, including restriction endonucleases).

One way in which the nucleic acid molecule may be linked to a ligand orsubstrate is through biotinylation. This can be done by PCRamplification with a 5′-biotinylation primer such that the biotin andnucleic acid are covalently linked.

The ligand or substrate to be selected can be attached to the modifiednucleic acid by a variety of means that will be apparent to those ofskill in the art. A biotinylated nucleic acid may be coupled to apolystyrene microbead (0.035 to 0.2 μm in diameter) that is coated withavidin or streptavidin, that will therefore bind the nucleic acid withvery high affinity. This bead can be derivatised with substrate orligand by any suitable method such as by adding biotinylated substrateor by covalent coupling.

Alternatively, a biotinylated nucleic acid may be coupled to avidin orstreptavidin complexed to a large protein molecule such as thyroglobulin(669 Kd) or ferritin (440 Kd). This complex can be derivatised withsubstrate or ligand, for example by covalent coupling to the ε-aminogroup of lysines or through a non-covalent interaction such asbiotin-avidin. The substrate may be present in a form unlinked to thegenetic element but containing an inactive “tag” that requires a furtherstep to activate it such as photoactivation (e.g. of a “caged” biotinanalogue, (Sundberg et al., 1995; Pirrung and Huang, 1996)). Thecatalyst to be selected then converts the substrate to product. The“tag” could then be activated and the “tagged” substrate and/or productbound by a tag-binding molecule (e.g. avidin or streptavidin) complexedwith the nucleic acid. The ratio of substrate to product attached to thenucleic acid via the “tag” will therefore reflect the ratio of thesubstrate and product in solution.

An alternative is to couple the nucleic acid to a product-specificantibody (or other product-specific molecule). In this scenario, thesubstrate (or one of the substrates) is present in each microcapsuleunlinked to the genetic element, but has a molecular “tag” (for examplebiotin, DIG or DNP). When the catalyst to be selected converts thesubstrate to product, the product retains the “tag” and is then capturedin the microcapsule by the product-specific antibody. In this way thegenetic element only becomes associated with the “tag” when it encodesor produces an enzyme capable of converting substrate to product.

When all reactions are stopped and the microcapsules are combined, thegenetic elements encoding active enzymes can be enriched using anantibody or other molecule which binds, or reacts specifically with the“tag”. Although both substrates and product have the molecular tag, onlythe genetic elements encoding active gene product will co-purify.

The terms “isolating”, “sorting” and “selecting”, as well as variationsthereof, are used herein. Isolation, according to the present invention,refers to the process of separating an entity from a heterogeneouspopulation, for example a mixture, such that it is free of at least onesubstance with which it was associated before the isolation process. Ina preferred embodiment, isolation refers to purification of an entityessentially to homogeneity. Sorting of an entity refers to the processof preferentially isolating desired entities over undesired entities. Inas far as this relates to isolation of the desired entities, the terms“isolating” and “sorting” are equivalent. The method of the presentinvention permits the sorting of desired genetic elements from pools(libraries or repertoires) of genetic elements which contain the desiredgenetic element. Selecting is used to refer to the process (includingthe sorting process) of isolating an entity according to a particularproperty thereof.

In a highly preferred application, the method of the present inventionis useful for sorting libraries of genetic elements. The inventionaccordingly provides a method according to preceding aspects of theinvention, wherein the genetic elements are isolated from a library ofgenetic elements encoding a repertoire of gene products. Herein, theterms “library”, “repertoire” and “pool” are used according to theirordinary signification in the art, such that a library of geneticelements encodes a repertoire of gene products. In general, librariesare constructed from pools of genetic elements and have properties whichfacilitate sorting.

Initial selection of a genetic element from a genetic element libraryusing the present invention will in most cases require the screening ofa large number of variant genetic elements. Libraries of geneticelements can be created in a variety of different ways, including thefollowing.

Pools of naturally occurring genetic elements can be cloned from genomicDNA or cDNA (Sambrook et al., 1989); for example, phage antibodylibraries, made by PCR amplification repertoires of antibody genes fromimmunised or unimmunised donors have proved very effective sources offunctional antibody fragments (Winter et al., 1994; Hoogenboom, 1997).Libraries of genes can also be made by encoding all (see for exampleSmith, 1985; Parmley and Smith, 1988) or part of genes (see for exampleLowman et al., 1991) or pools of genes (see for example Nissim et al.,1994) by a randomised or doped synthetic oligonucleotide. Libraries canalso be made by introducing mutations into a genetic element or pool ofgenetic elements ‘randomly’ by a variety of techniques in vivo,including; using ‘mutator strains’, of bacteria such as E. coli mutD5(Liao et al., 1986; Yamagishi et al., 1990; Low et al., 1996); using theantibody hypermutation system of B-ymphocytes (Yelamos et al., 1995).Random mutations can also be introduced both in vivo and in vitro bychemical mutagens, and ionising or UV irradiation (see Friedberg et al.,1995), or incorporation of mutagenic base analogues (Freese, 1959;Zaccolo et al., 1996). ‘Random’ mutations can also be introduced intogenes in vitro during polymerisation for example by using error-pronepolymerases (Leung et al., 1989).

Further diversification can be introduced by using homologousrecombination either in vivo (see Kowalczykowski et al., 1994 or invitro (Stemmer, 1994a; Stemmer, 1994b)).

According to a further aspect of the present invention, therefore, thereis provided a method of in vitro evolution comprising the steps of:

-   -   (a) selecting one or more genetic elements from a genetic        element library according to the present invention;    -   (b) mutating the selected genetic element(s) in order to        generate a further library of genetic elements encoding a        repertoire to gene products; and    -   (c) iteratively repeating steps (a) and (b) in order to obtain a        gene product.with enhanced activity.

Mutations may be introduced into the genetic elements(s) as set forthabove.

The genetic elements according to the invention advantageously encodeenzymes, preferably of pharmacological or industrial interest,activators or inhibitors, especially of biological systems, such ascellular signal transduction mechanisms, antibodies and fragmentsthereof, other binding agents suitable for diagnostic and therapeuticapplications. In a preferred aspect, therefore, the invention permitsthe identification and isolation of clinically or industrially usefulproducts. In a further aspect of the invention, there is provided aproduct when isolated by the method of the invention.

The selection of suitable encapsulation conditions is desirable.Depending on the complexity and size of the library to be screened, itmay be beneficial to set up the encapsulation procedure such that 1 orless than 1 genetic element is encapsulated per microcapsule. This willprovide the greatest power of resolution. Where the library is largerand/or more complex, however, this may be impracticable; it may bepreferable to encapsulate several genetic elements together and rely onrepeated application of the method of the invention to achieve sortingof the desired activity. A combination of encapsulation procedures maybe used to obtain the desired enrichment.

Theoretical studies indicate that the larger the number of geneticelement variants created the more likely it is that a molecule will becreated with the properties desired (see Perelson and Oster, 1979 for adescription of how this applies to repertoires of antibodies). Recentlyit has also been confirmed practically that larger phage-antibodyrepertoires do indeed give rise to more antibodies with better bindingaffinities than smaller repertoires (Griffiths et al., 1994). To ensurethat rare variants are generated and thus are capable of being selected,a large library size is desirable. Thus, the use of optimally smallmicrocapsules is beneficial.

The largest repertoire created to date using methods that require an invivo step (phage-display and LacI systems) has been a 1.6×10¹¹ clonephage-peptide library which required the fermentation of 15 litres ofbacteria (Fisch et al., 1996). SELEX experiments are often carried outon very large numbers of variants (up to 10¹⁵).

Using the present invention, at a preferred microcapsule diameter of 2.6μm, a repertoire size of at least 10¹¹ can be selected using 1 mlaqueous phase in a 20 ml emulsion.

In addition to the genetic elements described above, the microcapsulesaccording to the invention will comprise further components required forthe sorting process to take place. Other components of the system willfor example comprise those necessary for transcription and/ortranslation of the genetic element. These are selected for therequirements of a specific system from the following; a suitable buffer,an in vitro transcription/replication system and/or an in vitrotranslation system containing all the necessary ingredients, enzymes andcofactors, RNA polymerase, nucleotides, nucleic acids (natural orsynthetic), transfer RNAs, ribosomes and amino acids, and the substratesof the reaction of interest in order to allow selection of the modifiedgene product.

A suitable buffer will be one in which all of the desired components ofthe biological system are active and will therefore depend upon therequirements of each specific reaction system. Buffers suitable forbiological and/or chemical reactions are known in the art and recipesprovided in various laboratory texts, such as Sambrook et al., 1989.

The in vitro translation system will usually comprise a cell extract,typically from bacteria (Zubay, 1973; Zubay, 1980; Lesley et al., 1991;Lesley, 1995), rabbit reticulocytes (Pelham and Jackson, 1976), or wheatgerm (Anderson et al., 1983). Many suitable systems are commerciallyavailable (for example from Promega) including some which will allowcoupled transcription/translation (all the bacterial systems and thereticulocyte and wheat germ TNT™ extract systems from Promega). Themixture of amino acids used may include synthetic amino acids ifdesired, to increase the possible number or variety of proteins producedin the library. This can be accomplished by charging tRNAs withartificial amino acids and using these tRNAs for the in vitrotranslation of the proteins to be selected (Ellman et al., 1991; Benner,1994; Mendel et al., 1995).

After each round of selection the enrichment of the pool of geneticelements for those encoding the molecules of interest can be assayed bynon-compartmentalised in vitro transcription/replication or coupledtranscription-translation reactions. The selected pool is cloned into asuitable plasmid vector and RNA or recombinant protein is produced fromthe individual clones for further purification and assay.

The invention moreover relates to a method for producing a gene product,once a genetic element encoding the gene product has been sorted by themethod of the invention. Clearly, the genetic element itself may bedirectly expressed by conventional means to produce the gene product.However, alternative techniques may be employed, as will be apparent tothose skilled in the art. For example, the genetic informationincorporated in the gene product may be incorporated into a suitableexpression vector, and expressed therefrom.

The invention also describes the use of conventional screeningtechniques to identify compounds which are capable of interacting withthe gene products identified by the first aspect of the invention. Inpreferred embodiments, gene product encoding nucleic acid is.incorporated into a vector, and introduced into suitable host cells toproduce transformed cell lines that express the gene product. Theresulting cell lines can then be produced for reproducible qualitativeand/or quantitative analysis of the effect(s) of potential drugsaffecting gene product function. Thus gene product expressing cells maybe employed for the identification of compounds, particularly smallmolecular weight compounds, which modulate the function of gene product.Thus host cells expressing gene product are useful for drug screeningand it is a further object of the present invention to provide a methodfor identifying compounds which modulate the activity of the geneproduct, said method comprising exposing cells containing heterologousDNA encoding gene product, wherein said cells produce functional geneproduct, to at least one compound or mixture of compounds or signalwhose ability to modulate the activity of said gene product is sought tobe determined, and thereafter monitoring said cells for changes causedby said modulation. Such an assay enables the identification ofmodulators, such as agonists, antagonists and allosteric modulators, ofthe gene product. As used herein, a compound or signal that modulatesthe activity of gene product refers to a compound that alters theactivity of gene product in such a way that the activity of gene productis different in the presence of the compound or signal (as compared tothe absence of said compound or signal).

Cell-based screening assays can be designed by constructing cell linesin which the expression of a reporter protein, i.e. an easily assayableprotein, such as b galactosidase, chloramphenicol acetyltransferase(CAT) or luciferase, is dependent on gene product. Such an assay enablesthe detection of compounds that directly modulate gene product function,such as compounds that antagonise gene product, or compounds thatinhibit or potentiate other cellular functions required for the activityof gene product.

The present invention also provides a method to exogenously affect geneproduct dependent processes occurring in cells. Recombinant gene productproducing host cells, e.g. mammalian cells, can be contacted with a testcompound, and the modulating effect(s) thereof can then be evaluated bycomparing the gene product-mediated response in the presence and absenceof test compound, or relating the gene product-mediated response of testcells, or control cells (i.e., cells that do not express gene product),to the presence of the compound.

In a further aspect, the invention relates to a method for optimising aproduction process which involves at least one step which is facilitatedby a polypeptide. For example, the step may be a catalytic step, whichis facilitated by an enzyme. Thus, the invention provides a method forpreparing a compound or compounds comprising the steps of:

-   -   (a) providing a synthesis protocol wherein at least one step is        facilitated by a polypeptide;    -   (b) preparing genetic elements encoding variants of the        polypeptide which facilitates this step;    -   (c) compartmentalising the genetic elements into microcapsules;    -   (d) expressing the genetic elements to produce their respective        gene products within the microcapsules;    -   (e) sorting the genetic elements which produce polypeptide gene        product(s) having the desired activity; and    -   (f) preparing the compound or compounds using the polypeptide        gene product identified in (e) to facilitate the relevant step        of the synthesis.

By means of the invention, enzymes involved in the preparation of acompound may be optimised by selection for optimal activity. Theprocedure involves the preparation of variants of the polypeptide to bescreened, which equate to a library of polypeptides as refereed toherein. The variants may be prepared in the same manner as the librariesdiscussed elsewhere herein.

(B) Selection Procedures

The system can be configured to select for RNA, DNA or protein geneproduct molecules with catalytic, regulatory or binding activity.

(i) Affinity Selection

In the case of selection for a gene product with affinity for a specificligand the genetic element may be linked to the gene product in themicrocapsule via the ligand. Only gene products with affinity for theligand will therefore bind to the genetic element itself and thereforeonly genetic elements that produce active product will be retained inthe selection step. In this embodiment, the genetic element will thuscomprise a nucleic acid encoding the gene product linked to a ligand forthe gene product.

In this embodiment, all the gene products to be selected contain aputative binding domain, which is to be selected for, and a commonfeature—a tag. The genetic element in each microcapsule is physicallylinked to the ligand. If the gene product produced from the geneticelement has affinity for the ligand, it will bind to it and becomephysically linked to the same genetic element that encoded it, resultingin the genetic element being ‘tagged’. At the end of the reaction, allof the microcapsules are combined, and all genetic elements and geneproducts pooled together in one environment. Genetic elements encodinggene products exhibiting the desired binding can be selected by affinitypurification using a molecule that specifically binds to, or reactsspecifically with, the “tag”.

In an alternative embodiment, genetic elements may be sorted on thebasis that the gene product, which binds to the ligand, merely hides theligand from, for example, further binding partners. In this eventuality,the genetic element, rather than being retained during an affinitypurification step, may be selectively eluted whilst other geneticelements are bound.

In an alternative embodiment, the invention provides a method accordingto the first aspect of the invention, wherein in step (b) the geneproducts bind to genetic elements encoding them. The gene productstogether with the attached genetic elements are then sorted as a resultof binding of a ligand to gene products having the desired activity. Forexample, all gene products can contain an invariant region which bindscovalently or non-covalently to the genetic element, and a second regionwhich is diversified so as to generate the desired binding activity.

Sorting by affinity is dependent on the presence of two members of abinding pair in such conditions that binding may occur. Any binding pairmay be used for this purpose. As used herein, the term binding pairrefers to any pair of molecules capable of binding to one another.Examples of binding pairs that may be used in the present inventioninclude an antigen and an antibody or fragment thereof capable ofbinding the antigen, the biotin-avidin/streptavidin pair (Savage et al.,1994), a calcium-dependent binding polypeptide and ligand thereof (e.g.calmodulin and a calmodulin-binding peptide (Stofko et al., 1992;Montigiani et al. ,1996)), pairs of polypeptides which assemble to forma leucine zipper (Tripet et al., 1996), histidines (typicallyhexahistidine peptides) and chelated Cu²⁺, Zn²⁺ and Ni²⁺, (e.g. Ni—NTA;Hochuli et al., 1987), RNA-binding and DNA-binding proteins (Klug, 1995)including those containing zinc-finger motifs (Klug and Schwabe, 1995)and DNA methyltransferases (Anderson, 1993), and their nucleic acidbinding sites.

(ii) Catalysis

When selection is for catalysis, the genetic element in eachmicrocapsule may comprise the substrate of the reaction. If the geneticelement encodes a gene product capable of acting as a catalyst, the geneproduct will catalyse the conversion of the substrate into the product.Therefore, at the end of the reaction the genetic element is physicallylinked to the product of the catalysed reaction. When the microcapsulesare combined and the reactants pooled, genetic elements encodingcatalytic molecules can be enriched by selecting for any propertyspecific to the product (FIG. 1).

For example, enrichment can be by affinity purification using a molecule(e.g. an antibody) that binds specifically to the product. Equally, thegene product may have the effect of modifying a nucleic acid componentof the genetic element, for example by methylation (or demethylation) ormutation of the nucleic acid, rendering it resistant to or susceptibleto attack by nucleases, such as restriction endonucleases.

Alternatively, selection may be performed indirectly by coupling a firstreaction to subsequent reactions that takes place in the samemicrocapsule. There are two general ways in which this may be performed.First, the product of the first reaction could be reacted with, or boundby, a molecule which does not react with the substrate of the firstreaction. A second, coupled reaction will only proceed in the presenceof the product of the first reaction. An active genetic element can thenbe purified by selection for the properties of the product of the secondreaction.

Alternatively, the product of the reaction being selected may be thesubstrate or cofactor for a second enzyme-catalysed reaction. The enzymeto catalyse the second reaction can either be translated in situ in themicrocapsules or incorporated in the reaction mixture prior tomicroencapsulation. Only when the first reaction proceeds will thecoupled enzyme generate a selectable product.

This concept of coupling can be elaborated to incorporate multipleenzymes, each using as a substrate the product of the previous reaction.This allows for selection of enzymes that will not react with animmobilised substrate. It can also be designed to give increasedsensitivity by signal amplification if a product of one reaction is acatalyst or a cofactor for a second reaction or series of reactionsleading to a selectable product (for example, see Johannsson and Bates,1988; Johannsson, 1991). Furthermore an enzyme cascade system can bebased on the production of an activator for an enzyme or the destructionof an enzyme inhibitor (see Mize et al., 1989). Coupling also has theadvantage that a common selection system can be used for a whole groupof enzymes which generate the same product and allows for the selectionof complicated chemical transformations that cannot be performed in asingle step.

Such a method of coupling thus enables the evolution of novel “metabolicpathways” in vitro in a stepwise fashion, selecting and improving firstone step and then the next. The selection strategy is based on the finalproduct of the pathway, so that all earlier steps can be evolvedindependently or sequentially without setting up a new selection systemfor each step of the reaction.

Expressed in an alternative manner, there is provided a method ofisolating one or more genetic elements encoding a gene product having adesired catalytic activity, comprising the steps of:

-   -   (1) expressing genetic elements to give their respective gene        products;    -   (2) allowing the gene products to catalyse conversion of a        substrate to a product, which may or may not be directly        selectable, in accordance with the desired activity;    -   (3) optionally coupling the first reaction to one or more        subsequent reactions, each reaction being modulated by the        product of the previous reactions, and leading to the creation        of a final, selectable product;    -   (4) linking the selectable product of catalysis to the genetic        elements by either:        -   a) coupling a substrate to the genetic elements in such a            way that the product remains associated with the genetic            elements, or        -   b) reacting or binding the selectable product to the genetic            elements by way of a suitable molecular “tag” attached to            the substrate which remains on the product, or        -   c) coupling the selectable product (but not the substrate)            to the genetic elements by means of a product-specific            reaction or interaction with the product; and    -   (5) selecting the product of catalysis, together with the        genetic element to which it is bound, either by means of a        specific reaction or interaction with the product, or by        affinity purification using a suitable molecular “tag” attached        to the product of catalysis, wherein steps (1) to (4) each        genetic element and respective gene product is contained within        a microcapsule.        (iii) Regulation

A similar system can be used to select for regulatory properties ofenzymes.

In the case of selection for a regulator molecule which acts as anactivator or inhibitor of a biochemical process, the components of thebiochemical process can either be translated in situ in eachmicrocapsule or can be incorporated in the reaction mixture prior tomicroencapsulation. If the genetic element being selected is to encodean activator, selection can be performed for the product of theregulated reaction, as described above in connection with catalysis. Ifan inhibitor is desired, selection can be for a chemical propertyspecific to the substrate of the regulated reaction.

There is therefore provided a method of sorting one or more geneticelements coding for a gene product exhibiting a desired regulatoryactivity, comprising the steps of:

-   -   (1) expressing genetic elements to give their respective gene        products;    -   (2) allowing the gene products to activate or inhibit a        biochemical reaction, or sequence of coupled reactions, in        accordance with the desired activity, in such a way as to allow        the generation or survival of a selectable molecule;    -   (3) linking the selectable molecule to the genetic elements        either by    -   a) having the selectable molecule, or the substrate from which        it derives, attached to the genetic elements, or    -   b) reacting or binding the selectable product to the genetic        elements, by way of a suitable molecular “tag” attached to the        substrate which remains on the product, or    -   c) coupling the product of catalysis (but not the substrate) to        the genetic elements, by means of a product-specific reaction or        interaction with the product;    -   (4) selecting the selectable product, together with the genetic        element to which it is bound, either by means of a specific        reaction or interaction with the selectable product, or by        affinity purification using a suitable molecular “tag” attached        to the product of catalysis. wherein steps (1) to (4) each        genetic element and respective gene product is contained within        a microcapsule.        (iv) Microcapsule Sorting

The invention provides for the sorting of intact microcapsules wherethis is enabled by the sorting techniques being employed. Microcapsulesmay be sorted as such when the change induced by the desired geneproduct either occurs or manifests itself at the surface of themicrocapsule or is detectable from outside the microcapsule. The changemay be caused by the direct action of the gene product, or indirect, inwhich a series of reactions, one or more of which involve the geneproduct having the desired activity leads to the change. For example,the microcapsule may be so configured that the gene product is displayedat its surface and thus accessible to reagents. Where the microcapsuleis a membranous microcapsule, the gene product may be targeted or maycause the targeting of a molecule to the membrane of the microcapsule.This can be achieved, for example, by employing a membrane localisationsequence, such as those derived from membrane proteins, which willfavour the incorporation of a fused or linked molecule into themicrocapsule membrane. Alternatively, where the microcapsule is formedby phase partitioning such as with water-in-oil emulsions, a moleculehaving parts which are more soluble in the extra-capsular phase willarrange themselves such that they are present at the boundary of themicrocapsule.

In a preferred aspect of the invention, however, microcapsule sorting isapplied to sorting systems which rely on a change in the opticalproperties of the microcapsule, for example absorption or emissioncharacteristics thereof, for example alteration in the opticalproperties of the microcapsule resulting from a reaction leading tochanges in absorbance, luminescence, phosphorescence or fluorescenceassociated with the microcapsule. All such properties are included inthe term “optical”. In such a case, microcapsules can be sorted byluminescence, fluorescence or phosphorescence activated sorting. In ahighly preferred embodiment, fluorescence activated sorting is employedto sort microcapsules in which the production of a gene product having adesired activity is accompanied by the production of a fluorescentmolecule in the cell. For example, the gene product itself may befluorescent, for example a fluorescent protein such as GFP.Alternatively, the gene product may induce or modify the fluorescence ofanother molecule, such as by binding to it or reacting with it.

(v) Microcapsule Identification

Microcapsules may be identified by virtue of a change induced by thedesired gene product which either occurs or manifests itself at thesurface of the microcapsule or is detectable from the outside asdescribed in section iii (Microcapsule Sorting). This change, whenidentified, is used to trigger the modification of the gene within thecompartment. In a preferred aspect of the invention, microcapsuleidentification relies on a change in the optical properties of themicrocapsule resulting from a reaction leading to luminescence,phosphorescence or fluorescence within the microcapsule. Modification ofthe gene within the microcapsules would be triggered by identificationof luminescence, phosphorescence or fluorescence. For example,identification of luminescence, phosphorescence or fluorescence cantrigger bombardment of the compartment with photons (or other particlesor waves) which leads to modification of the genetic element. A similarprocedure has been described previously for the rapid sorting of cells(Keij et al., 1994). Modification of the genetic element may result, forexample, from coupling a molecular “tag”, caged by a photolabileprotecting group to the genetic elements: bombardment with photons of anappropriate wavelength leads to the removal of the cage. Afterwards, allmicrocapsules are combined and the genetic elements pooled together inone environment. Genetic elements encoding gene products exhibiting thedesired activity can be selected by affinity purification using amolecule that specifically binds to, or reacts specifically with, the“tag”.

(vi) Multi-Step Procedure

It will be also be appreciated that according to the present invention,it is not necessary for all the processes of transcription/replicationand/or translation, and selection to proceed in one single step, withall reactions taking place in one microcapsule. The selection proceduremay comprise two or more steps. First, transcription/replication and/ortranslation of each genetic element of a genetic element library maytake place in a first microcapsule. Each gene product is then linked tothe genetic element which encoded it (which resides in the samemicrocapsule). The microcapsules are then broken, and the geneticelements attached to their respective gene products optionally purified.Alternatively, genetic elements can be attached to their respective geneproducts using methods which do not rely on encapsulation. For examplephage display (Smith, G. P.,1985), polysome display (Mattheakkis et al.,1994), RNA-peptide fusion (Roberts and Szostak, 1997) or lac repressorpeptide fusion (Cull, et al., 1992).

In the second step of the procedure, each purified genetic elementattached to its gene product is put into a second microcapsulecontaining components of the reaction to be selected. This reaction isthen initiated. After completion of the reactions, the microcapsules areagain broken and the modified genetic elements are selected. In the caseof complicated multistep reactions in which many individual componentsand reaction steps are involved, one or more intervening steps may beperformed between the initial step of creation and linking of geneproduct to genetic element, and the final step of generating theselectable change in the genetic element.

(vii) Selection by Activation of Reporter Gene Expression in Situ

The system can be configured such that the desired binding, catalytic orregulatory activity encoded by a genetic element leads, directly orindirectly to the activation of expression of a “reporter gene” that ispresent in all microcapsules. Only gene products with the desiredactivity activate expression of the reporter gene. The activityresulting from reporter gene expression allows the selection of thegenetic element (or of the compartment containing it) by any of themethods described herein.

For example, activation of the reporter gene may be the result of abinding activity of the gene product in a manner analogous to the “twohybrid system” (Fields and Song, 1989). Activation might also resultfrom the product of a reaction catalysed by a desirable gene product.For example, the reaction product could be a transcriptional inducer ofthe reporter gene. For example arabinose could be used to inducetranscription from the araBAD promoter. The activity of the desirablegene product could also result in the modification of a transcriptionfactor, resulting in expression of the reporter gene. For example, ifthe desired gene product is a kinase or phosphatase the phosphorylationor dephosphorylation of a transcription factor may lead to activation ofreporter gene expression.

(viii) Amplification

According to a further aspect of the present invention the methodcomprises the further step of amplifying the genetic elements. Selectiveamplification may be used as a means to enrich for genetic elementsencoding the desired gene product.

In all the above configurations, genetic material comprised in thegenetic elements may be amplified and the process repeated in iterativesteps. Amplification may be by the polymerase chain reaction (Saiki etal., 1988) or by using one of a variety of other gene amplificationtechniques including; Qβ replicase amplification (Cahill, Foster andMahan, 1991; Chetverin and Spirin, 1995; Katanaev, Kumasov and Spirin,1995); the ligase chain reaction (LCR) (Landegren et al., 1988; Barany,1991); the self-sustained sequence replication system (Fahy, Kwoh andGingeras, 1991) and strand displacement amplification (Walker et al.,1992).

(ix) Compartmentalisation

According to a further aspect of the present invention, there isprovided a method for compartmentalising a genetic element andexpressing the genetic element to form its gene product within thecompartment, comprising the steps of:

-   -   (a) forming an aqueous solution comprising the genetic element        and the components necessary to express it to form its gene        product;    -   (b) microencapsulating the solution so as to form a discrete        microcapsule comprising the genetic element; and    -   (c) exposing the microcapsule to conditions suitable for the        expression of the genetic element to form its gene product to        proceed.

Suitable microencapsulation techniques are described in detail in theforegoing general description.

Preferably, a library of genetic elements encoding a repertoire of geneproducts is encapsulated by the method set forth above, and the geneticelements expressed to produce their respective gene products, inaccordance with the invention. In a highly preferred embodiment,microencapsulation is achieved by forming a water-in-oil emulsion of theaqueous solution comprising the genetic elements.

The invention, accordingly, also provides a microcapsule obtainable bythe method set forth above.

Various aspects and embodiments of the present invention are illustratedin the following examples. It will be appreciated that modification ofdetail may be made without departing from the scope of the invention.

All documents mentioned in the text are incorporated by reference.

EXAMPLES Example 1

The Production of Approx. 2 μm Aqueous Microcapsules in a Water-in-oilEmulsion System.

Microcapsules within the preferred size range of the present inventioncan be generated using a water-in-oil emulsion system.

Light white mineral oil (Sigma; M-3516) is used herein as the continuousphase and the emulsion is stabilised by emulsifiers sorbitan monooleate(Span 80, Fluka; 85548) and polyoxyethylenesorbitan monooleate (Tween80, Sigma Ultra; P-8074) and in some cases also with 0.5% w/v sodiumdeoxycholate (Fluka; 30970).

The oil phase is freshly prepared by dissolving 4.5% (v/v) Span 80(Fluka) in mineral oil (Sigma, #M-5904) followed by 0.5% (v/v) Tween 80(SigmaUltra; #P-8074). Ice-cooled in vitro reaction mixtures (50 μl) areadded gradually (in 5 aliquots of 10 μl over ˜2 minutes) to 0.95 ml ofice-cooled oil-phase in a 5 ml Costar Biofreeze Vial (#2051) whilststirring with a-magnetic bar (8×3 mm with a pivot ring; ScientificIndustries International, Loughborough, UK). Stirring (at 1150 rpm) iscontinued for an additional 1 minute on ice. In some emulsions theaqueous phase is supplemented with an anionic surfactant—e.g., sodiumdeoxycholate, sodium cholate, sodium glycocholate, and sodiumtaurocholate, typically to 0.5% (w/v).

When indicated, the emulsion is further homogenised using anUltra-Turrax T25 disperser (IKA) equipped with an 8 mm diameterdispersing tool at 8 k, 9 k or 13.5 k rpm for 1 minute, or at 20 k rpmfor 1 or 5 minutes, on ice. This reduces the microcapsule size.

The reactions may be quenched and the emulsion broken as indicated inindividual examples, by spinning at 3,000 g for 5 minutes and removingthe oil phase, leaving the concentrated emulsion at the bottom of thevial. Quenching buffer (typically, 0.2 ml of 25 μg/ml yeast RNA in W+Bbuffer: 1 M NaCl, 10 mM Tris-HCl, 1 mM EDTA pH 7.4) and 2 ml ofwater-saturated diethyl ether is added and the mixture vortexed,centrifuged briefly, and the ether phase removed. The aqueous phase iswashed with ether and dried (5 minutes in a Speedvac at ambienttemperature).

The size distribution of the aqueous droplets in the emulsions wasdetermined by laser diffraction using a Coulter LS230 Particle SizeAnalyser. An aliquot of emulsion, freshly diluted (1:10) in mineral oilis added to the micro-volume chamber containing stirred mineral oil.Results are analysed with the instrument's built-in Mie optical modelusing refractive indices of 1.468 for mineral oil and 1.350 for theaqueous phase. The size distribution of the aqueous droplets in theemulsion is shown in FIG. 2. Addition of sodium deoxycholate does notsignificantly alter the size distribution.

Example 2

Efficient in vitro Transcription Reactions Performed in the AqueousMicrocapsules of a Water-in-oil Emulsion.

In order to produce RNA from DNA within each microcapsule, the singlemolecule of DNA present within each aqueous microcapsule of the systemmust be transcribed efficiently. Herein, in vitro transcription isdemonstrated within microcapsules.

The catalytic core of the Tetrahymena self-splicing intron is amuch-studied ribozyme which can catalyse a variety of phosphoestertransfer reactions (Sag et al., 1986; Sag and Czech, 1986; Sag andCzech, 1986). For example, a modified Tetrahymena intron missing the P1stem-loop from the 5′-end, and missing the 3′ stem-loops P9.1 and P9.2can function as an RNA ligase, efficiently splicing together two or moreoligonucleotides aligned on a template strand (Green and Szostak, 1992).

DNA encoding the above-described Tetrahymena ribozyme is PCR-amplifiedusing primers P2T7Ba (which anneals to the P2 loop region and appends aT7 RNA polymerase promoter) and P9Fo (which anneals to the P9 loopregion). This creates a 331 base pair DNA fragment carrying the T7 RNApolymerase promoter. This fragment is purified directly using Wizard PCRPreps (Promega) and used as the template for an in vitro transcriptionreaction using T7 RNA polymerase.

In vitro transcription is assayed over an initial 10 minute periodduring which the reaction rate is essentially linear (Chamberlin andRing, 1973). Reaction conditions for transcription are as described byWyatt et al., 1991.

Incorporation of [γ⁻³²P] UTP is used to assay the progression of thereaction.

A transcription reaction is set up in a volume of 200 μl and dividedinto 2 aliquots, each containing 3×10¹¹ molecules of DNA (5 nM). One 100μl aliquot is added to 2 ml Sigma light mineral oil containing 4.5% Span80 and 0.5% Tween 80 and homogenised for 5 minutes with an Ultra-TurraxT25 disperser at 20,000 rpm as in Example 1. Based on the meanmicrocapsule volume in these emulsions (2.8×10⁻¹⁹ m³ for a 0.81 μmdiameter microcapsule) the 100 μl reaction would be divided into3.6×10¹¹ microcapsules. Hence, there should be 1 molecule of DNA permicrocapsule on average.

Both aliquots are incubated in a 37° C. water bath. 0.5 ml samples ofthe emulsion are removed both before the start of the incubation andafter 10 minutes and placed on ice. Similar 25 μl samples are removedfrom the non-emulsified control reactions at the same time. Emulsionsare broken and reactions stopped with 0.5 ml EDTA (50 mM) and 2 mlwater-saturated diethyl ether as described in Example 1. 100 μl salmonsperm DNA (500 μg/ml) in 20 mM EDTA is then added. Three 100 μl aliquotsare then removed from both emulsions and controls and labelled RNA isassayed by TCA precipitation and scintillation counting.

The rate of transcription is taken as the increase in acid perceptiblecpm over the 10 minute incubation at 37° C. In the non emulsifiedcontrol reaction there are 442,000 cpm acid perceptible materialcompared to 147,000 cpm in the emulsion. Hence the rate of transcriptionin the emulsion is 33% of that found in the non-emulsified controlreaction.

This procedure therefore shows that RNA can be efficiently synthesisedby T7 RNA polymerase in the aqueous microcapsules of a water-in-oilemulsion.

Example 3

Efficient Coupled in vitro Transcription/Translation Reactions Performedin the Aqueous Microcapsules of a Water-in-oil Emulsion.

In order to synthesise proteins using the procedure of the presentinvention, translation must be active in the aqueous microcapsules ofthe water-in-oil emulsion described herein.

Here it is shown how a protein (E. coli dihydrofolate reductase) can beefficiently produced from DNA in the aqueous microcapsules of awater-in-oil emulsion system using a coupled transcription/translationsystem.

The E. coli folA gene encoding dihydrofolate reductase (DHFR) isPCR-amplified using oligonucleotides EDHFRFo and EDHFRBa. This DNA isthen cloned into the pGEM-4Z vector (Promega) digested with HindIII andKpnI downstream of the both the lac promoter and the T7 RNA polymerasepromoter. The oligonucleotide EDHFRBa appends the efficient phage T7gene 10 translational start site upstream of the DHFR start codon.

DNA sequencing identifies a clone which has the correct nucleotidesequence. Bacteria transformed with this clone (pGEM-folA) are found toover express active DHFR (driven from the lac promoter) when inducedwith IPTG.

The pGEM-folA plasmid is then PCR-amplified using primers LMB2 and LMB3under the conditions described above to create a 649 bp DNA fragmentcarrying the T7 RNA polymerase promoter, the phage T7 gene 10translational start site and the folA gene. This PCR fragment ispurified directly using Wizard PCR Preps (Promega) and used to program aprokaryotic in vitro coupled transcription/translation system designedfor linear templates (Lesley, Brow and Burgess, 1991).

A commercial preparation of this system is used (E. coli S30 ExtractSystem for Linear Templates; Promega) supplemented with T7 RNApolymerase.

A 300 μl translation reaction is set up on ice containing 3×10¹²molecules of DNA. T7 RNA polymerase (10⁴ units) is added to drivetranscription and the translated protein is labelled by the addition of[³⁵S] methionine. A 150 μl aliquot of this reaction is added to 2.85 mlSigma light mineral oil containing 4.5% Span 80 and 0.5% Tween 80 andhomogenised for 1 minute with an Ultra-Turrax T25 disperser at 20,000rpm, as in Example 1. The other aliquot is not emulsified.

Based on the mean microcapsule volume in the emulsions (1.1×10⁻¹⁸ m³ fora 1.29 μm diameter microcapsule) the 150 μl reaction would be dividedinto 1.3×10¹¹, microcapsules). Hence, there should be roughly 11molecules of DNA per microcapsule.

Four 0.5 ml aliquots are removed from the emulsion reaction mix. Onealiquot is immediately put on ice and the other three are incubated in a25° C. water bath for 2 hours before being placed on ice. Four 25 μlsamples are also removed from the non-emulsified reaction mix; one isput immediately on ice and the other three are incubated in a 25° C.water bath for 2 hours and then placed on ice.

The emulsions are spun in a microfuge at 13,000 r.p.m. for 5 minutes at4° C. and the mineral oil removed leaving the concentrated (but stillintact) emulsion at the bottom of the tube. After briefly re-spinningand removing any further mineral oil, the emulsion is broken and anyfurther translation stopped by adding 100 μl water containing 125 μg/mlpuromycin, and 1 ml water saturated diethyl ether. This mixture isvortexed and respun in a microfuge at 13,000 r.p.m. for 1 minute at 4°C. The ether and dissolved mineral oil is then removed by aspiration andthe extraction repeated with a further 1 ml of ether. Any remainingether is driven off by spinning for 5 minutes in a Speedvac at roomtemperature.

100 μl water containing 125 μg/ml puromycin is also added to the 25 μlnon-emulsified control reactions. 25 μl of each of the samples is thenprecipitated with acetone and run on a 20% SDS-PAGE gel according to theinstructions given by the manufacturers of the in vitrotranscription/translation system (Promega). The gel is dried and scannedusing a PHOSPHORIMAGER™ (quantitative imaging device; MOLECULARDYNAMICS). A single strong band is seen with the expected molecularweight of DHFR (18 kd) in both the reactions performed in emulsions andin the controls. This band is accurately quantified.

In the emulsified reactions the mean area under the 18 kd peak is 15,073units whereas the mean area under the same peak in the non-emulsifiedcontrol reactions is 18,990 units. Hence, in the emulsified reactionsthe amount of DHFR protein is calculated to be 79% that found in thenonemulsified control reactions. This therefore indicates that thetranscription/translation system is functional in the water-in-oilemulsion system of the present invention.

Example 4

Dihydrofolate Reductase Produced Using the Coupled in vitroTranscription/Translation Reactions is Active.

Here it is shown that protein (E. coli dihydrofolate reductase) can beproduced efficiently in a catalytically active form by coupledtranscription/translation of the folA gene in the aqueous microcapsulesof a water-in-oil emulsion system. In this assay, an emulsion comprisingmicrocapsules below optimal size is used; DHFR activity is shown to behigher in the larger microcapsule sizes.

175 μl translation reactions (unlabelled) are set up on ice containingeither 2×10¹¹, 6×10¹² or 1.8×10¹² molecules of the folA template DNAused in Example 3, or no DNA. T7 RNA polymerase (6×10³ units) are addedto each reaction to drive transcription.

A 100 μl aliquot of each reaction is added to 1.9ml Sigma light mineraloil containing 4.5% Span 80 and 0.5% Tween 80 and homogenised for 1minute or 5 minutes with an Ultra-Turrax T25 Homogeniser equipped withan 8 mm diameter dispersing tool, at 20,000 rpm as in Example 1. Afterhomogenisation for 1 minute the mean diameter of particles (by volume)is 1.30 μm (median 1.28 μm). 98% by volume of the internal (aqueous)phase is present in particles varying from 0.63 μm to 2.12 μm. Afterhomonogenisation for 5 minutes the mean diameter of microcapsules (byvolume) is 0.81 μm (median 0.79 μm) and 98% by volume of the internal(aqueous) phase is present in particles varying from 0.41 μm to 1.38 μM.

Based on the mean microcapsule volume in the 1 minute emulsions(1.1×10⁻¹⁸ m³ for a 1.299 μm diameter microcapsule) the 100 μl reactionwould be divided into 8.7×10¹⁰ nicrocapsules). Hence, there should beroughly 1.3, 3.9 or 11.8 molecules of DNA per microcapsule.

Based on the mean microcapsule volume in the 5 min emulsions (2.8×10−¹⁹M³ for a 0.81 μm diameter microcapsule) the 100 μl reaction would bedivided into 3.6×10¹¹ microcapsules). Hence, there should be roughly0.3, 1.0 or 2.9 molecules of DNA per microcapsule.

The emulsions, and the non-emulsified reaction mix are incubated in a25° C. water bath. 0.5 ml samples of the emulsion are removedimmediately before the start of the incubation and after 2 hours andplaced on ice. 25 μl samples are removed from the non-emulsified controlreactions at the same times.

The emulsions are spun in a microfuge at 13,000 r.p.m. for 5 min. at 4°C. and the mineral oil removed by aspiration, leaving the concentrated(but still intact) emulsion at the bottom of the tube. After brieflyre-spinning and removing any further mineral oil the emulsion is brokenand any further translation stopped by adding 100 μl Buffer A (100 mMImidazole pH 7.0, 10 mM β-mercaptoethanol), containing 125 μg/mlpuromycin and 1 ml water saturated diethyl ether. The mixture isvortexed and spun in a microfuge at 13,000 r.p.m. for 1 min. at 4° C.The ether and dissolved mineral oil is removed by aspiration and theextraction repeated with a further 1 ml of ether. Any remaining ether isdriven off by spinning for 5 minutes in a Speedvac at room temperature.100 μl Buffer A containing (125 μg/ml) puromycin is also added to the 25μl non-emulsified control reactions.

Dihydrofolate reductase activity is assayed as by spectrophotometricallymonitoring the oxidation of NADPH to NADP at 340 nm over a 10 minutetime course as described by Williams et al., 1979; Ma et al., 1993. 10μl of each quenched in vitro translation reaction is added to 150 μlBuffer A (100 mM Imidazole, pH 7.0, 10 mM β-mercaptoethanol) and 20 μl 1mM NADPH. 20 μl Dihydrofolate (1 mM)(H₂F) is added after 1 minute andthe reaction monitored at 340 nm using a ThermoMax microplate reader(Molecular Devices). Activity is calculated by initial velocities underSo>>K_(M) conditions (υ_(max)) The background activity in the S30extract is subtracted from all samples.

DHFR activity generated in the emulsions is taken from the difference inactivity measured at 0 hours and 2 hours incubation. No increase inNADPH oxidation occurred between the 0 hour and 2 hour samples when 0.1μM methotrexate (a specific inhibitor of DHFR) is added showing that allthe increase in NADPH oxidation observed is due to DHFR produced in thein vitro translation reactions.

Using 1 minute homogenisation at 20,000 rpm, the DHFR activity generatedin the emulsions is 31% that found in the non-emulsified controlreactions with 1.3 molecules of DNA per microcapsule; 45% with 3.9molecules of DNA per microcapsule; and 84% with 11.8 molecules of DNAper microcapsule.

Using 5 minute homogenisation at 20,000 rpm, the DHFR activity generatedin the emulsions is 7% that found in the non-emulsified controlreactions with 0.3 molecules of DNA per microcapsule; 15% with 1molecule of DNA per microcapsule; and 35% with 2.9 molecules of DNA permicrocapsule, on average.

Assuming the turnover number of DHFR is as described by Posner et al.,1996, this corresponds to a yield at the highest DNA concentration of6.3 μg (340 pmole) DHFR per 100 μl reaction (non-emulsified control),1.98 μg (104 pmole) DHFR per 100 μl reaction (emulsified for 1 min), or0.46 μg (24.8 pmole) per 100 μl reaction (emulsified for 5 minutes).This equates to 74 molecules DHFR per microcapsule in the 1 minuteemulsions and 44 molecules per microcapsule in the 5 minute emulsions(assuming that all microcapsules are of mean size).

The DHFR activity resulting from coupled transcription/translation offolA genes is also measured in the larger microcapsules produced bystirring alone, or by stirring followed by further homogenisation withan Ultra-Turrax T25 disperser at 8,000 rpm, 9,000 rpm, or 13,500 rpm for1 minute as described in Example 1. The results are presented in FIG. 2b. The concentration of folA genes used (2.5 nM) gives an average of 1,1.5 and 4.8 genetic elements per droplet in the emulsions homogenised at13,500 rpm, 9,500 rpm and 8,000 rpm, respectively, and an average of 14genetic element per droplet in the emulsion prepared by stirring only.Addition of sodium deoxycholate (0.5%) to the in vitro translationreaction mixture does not significantly affect the DHFR activityobserved in the broken emulsions.

Example 5

Linkage of an Immobilised Substrate into a Genetic Element via a HighMolecular Weight Protein.

In order to link multiple immobilised substrate molecules to a DNAfragment comprising the folA gene, the DNA fragment is firstbiotinylated and then coupled to a complex of avidin with apoferritin.Horse spleen apoferritin is a large, near spherical protein molecule of12.5 nm diameter which therefore provides multiple sites which can bederivatised with substrate (e.g. the ε-amino group of surface lysines).The pGEM-folA plasmid encoding E. coli DHFR is PCR amplified using theprimers LMB3 and 5′-biotinylated LMB2 (LMB2-Biotin) to create abiotinylated 649 bp DNA fragment carrying the T7 RNA polymerasepromoter, the phage T7 gene 10 translational start site and the folAgene (see Example 3). The DNA is radiolabelled by supplementing the 500μl PCR reaction mix with 100 μCi [α-32P]dCTP (Amersham; 3000 Ci/mmol).The biotinylated PCR fragment is purified directly using WIZARD™ PCRPreps (PCR purification kit; PROMEGA) and the concentration determinedspectrophotometrically. The percentage of DNA biotinylated is assayed bybinding to Streptavidin M-280 DYNABEADS™ (superparamagnetic particles;DYNAL) and scintillation counting. 83% of the DNA is determined to bebiotinylated using this technique.

The sequestered iron is removed from a commercial conjugate of avidinand ferritin (Avidin-Ferritin; approx. 1.1 mole ferritin per moleavidin; Sigma) by the overnight dialysis (4° C.) of a solution ofavidin-ferritin in PBS (1 mg/ml) against 0.12M thioglycollic acid, pH4.25, followed by 24 hours dialysis against PBS (4° C.) as described byKadir and Moore, 1990. Removal of iron is checked by analysis of theabsorbance spectra (sequestered Fe(III) absorbs strongly at 310-360 nm).

0.3 pmole radiolabelled, biotinylated DNA is incubated with varyingmolar ratios of avidin-apoferritin in PBS (total volume 9 μl) for 30minutes at room temperature. A 4.5 μl aliquot is removed and thepercentage of DNA complexed with avidin-apoferritin assayed usingband-shifting assay on a 1.5% agarose gel as described by Berman et al.,1987. The gel is then dried and scanned using a PHOSPHORIMAGER™(quantitative imaging device; MOLECULAR DYNAMICS). The percentage of DNAremaining unshifted (i.e. not complexed with avidin-apoferritin) is 17%(1:1 molar ratio avidin-apoferritin:DNA), 15% (5:1 molar ratioavidin-apoferritin:DNA) or 14% (25:1 molar ratioavidin-apoferritin:DNA). This means that even at a 1:1 ratio ofavidin-apoferritin:;DNA basically all the biotinylated DNA is bound. Noband-shifting is observed when biotinylated DNA is mixed withapoferritin or when non-biotinylated DNA is mixed withavidin-apoferritin.

The remaining 4.5 μl of DNA complexed with avidin-apoferritin is used asthe template for a 25 μl in vitro transcription/translation reaction (E.coli S30 Extract System for Linear Templates; Promega). After 2 hours at25° C., the reaction is stopped by adding 100 μl Buffer A containingpuromycin (125 μg/ml). Dihydrofolate reductase activity is assayed asabove by spectrophotometrically monitoring the oxidation of NADPH toNADP at 340 nm over a 10 minute time course.

10 μl of each in vitro translation reaction is added to 150 μl Buffer Aand 20 μl NADPH (1 mM). 20 μl Dihydrofolate (1 mM) (Emulsions werebroken and reactions were stopped with 0.5 ml EDTA (50 mM) and 2 mlwater-saturated diethyl ether as described in Example 1) is added after1 minute and the reaction monitored at 340 nm using a ThermoMaxmicroplate reader (Molecular Devices). No difference in DHFR activity isfound at even the highest ratio avidin-apoferritin:DNA compared to acontrol with no avidin-apoferritin added. This indicates that the vastmajority of DNA can be complexed without compromising the efficiency ofin vitro translation.

Example 6

Both in vitro Transcription-translation and DHFR Activity are Compatiblein the Same System.

In order to select for the activity of DHFR produced in situ by coupledtranscription-translation both the transcription-translation reactionand DHFR must be active in the same buffer system.

A direct assay for DHFR activity in a complete E. coli in vitrotranslation system based on the spectrophotometrically monitoring of theoxidation of NADPH to NADP at 340 nm is not practical due to theturbidity of the S30 extracts.

However, it is possible to ascertain that DHFR is active in the samebuffer system as in vitro translation. E. coli DHFR is obtained byIPTG-induction of bacteria containing the plasmid pGEM-folA andaffinity-purified on a methotrexate-Sepharose column (Baccanari et al.,1977).

DHFR activity is compared in Buffer A as above or in an in vitrotranslation mixture complete except for the substitution of S30 dialysisbuffer (Lesley 1995) (10 mM Tris-acetate pH8.0, 14 mM magnesium acetate,60 mM potassium acetate, 1 mM DTT) for the S30 fraction. In each casethe total reaction volume is 200 μl and the concentration of NADPH andEmulsions were broken and reactions were stopped with 0.5 ml EDTA (50mM) and 2 ml water-saturated diethyl ether as described in Example 1each 0.1 mM. Reactions are monitored spectrophotometrically at 340 nm.Addition of 1.75 pmole (1.3 mUnits) E. coli DHFR gives initial rates of−25.77 mOD/min (in Buffer A) and −11.24 mOD/min (in translation buffer),hence the reaction is 44% as efficient in the translation buffer as inan optimised buffer (buffer A).

Furthermore, the presence of the substrates of DHFR (NADPH and H₂F) at0.1 mM concentration (either alone or in combination) does not cause anyinhibition of the production of active DHFR from a 2 hour coupledtranscription-translation reaction.

Example 7

The Activity of DHFR on a Genetic Element Containing an ImmobilisedDihydrofolate Substrate Leads to the Formation of a TetrahydrofolateProduct Linked to Nucleic Acid Encoding DHFR.

A peptide is synthesised comprising three glutamic acids linked viatheir γ-caboxylates (using N-fluorenylmethoxycarbonyl-glutamic acidα-benzyl ester as a starting material) with a lysine at thecarboxy-terminus and biotin linked to its ε-amino group by modifyingpublished procedures (Krumdiek et al., 1980). Folic acid is linked atthe amino-terminus and the benzyl and trifluoroacetamide protectivegroups removed by alkaline hydrolysis as previously described. Thepeptide is purified by reverse phase HPLC and characterised by mass andUV spectroscopy. This folic acid peptide is chemically reduced to thecorresponding dihydrofolic acid peptide (using dithionate and ascorbicacid) and then to the corresponding tetrahydrofolic acid peptide (usingsodium borohydride) by applying published procedures (Zakrzewski et al.,1980). These transformations are characterised by UV spectroscopy.

A genetic element is constructed by linking, on average, two to threemolecules of the folic acid peptide to avidin (or streptavidin) togetherwith one molecule of the DHFR encoding, PCR-amplified DNA from theplasmid pGEM-folA using primers LMB2-Biotin (SEQ. ID. No. 9) and LMB3(see Example 3). The immobilised folic acid is chemically reduced todihydrofolate using dithionate and ascorbic acid and purified bydialysis against buffer A. E. coli DHFR is obtained by IPTG induction ofbacteria containing the plasmid pGEM-folA and affinity purified on amethotrexate-Sepharose column. E. coli DHFR is shown to react with thedihydrofolic acid immobilised to this genetic element by monitoring theoxidation of NADPH to NADP spectrophotometrically using 0-10 μM of theavidin-linked dihydrofolic acid peptide and 0-50 μM NADPH. Hence, at theend of this reaction, the product tetrahydrofolate is linked to the folAgene which encodes for the enzyme (i.e., DHFR) that catalyses itsformation.

To isolate those genes attached to the tetrahydrofolate product thereare two approaches. The first involves the generation of phage-displayantibodies specific for tetrahydrofolate (Hoogenboom, 1997). The secondapproach is based on the use of a tagged reagent which reactsspecifically with the immobilised product, but not with the substrate.We have synthesised a molecule consisting of a dinitrophenyl (DNP) taglinked to benzaldehyde via a 14 atom spacer. The aldehyde group reactsspecifically with tetrahydrofolate to form a covalent adduct (Kallen andJencks, 1966) and affinity purification can be performed using ananti-DNP antibody.

Example 8

An Alternative Method of Selecting for DHFR Activity

The DHFR-catalysed reaction can be selected for by in situ coupling to asecond reaction, catalysed by Yeast Aldehyde Dehydrogenase, with a‘tagged’ substrate.

Instead of selecting for genes connected to one of the products of theDHFR reaction (5,6,7,8-tetrahydrofolate or NADP+) the DHFR reaction iscoupled to a second reaction. Selection is in this case is mediated bythe formation of the second product of the DHFR-catalysedreaction—nicotinamide adenine dinucleotide phosphate (NADP+).

The reaction we have chosen to couple is catalysed by Yeast AldehydeDehydrogenase (YAD; EC 1.2.1.5). This enzyme uses either NAD+ or NADP+in the oxidation of a wide range of aliphatic and aromatic aldehydes totheir corresponding carboxylic acids, generating NADH or NADPH in theprocess. The reaction has the big advantage of being essentiallyirreversible—namely, dehydrogenases (including DHFR and YAD) do notcatalyse the reduction of the acid back to the aldehyde. Since a largenumber of enzymes catalysing redox reactions generate NAD+ or NADP+ theYAD reaction can be used in the selection of these enzymes too, and isnot limited solely to selection for Dihydrofolate Reductase.

A pentaldehyde substrate is synthesised and linked to a DNP(dinitrophenyl) tag via a C₂₀ linker (hereafter, DNP-PA). The oxidationof DNP-PA to the corresponding acid (DNP-PC) is followed and by HPLC(reverse phase C₁₈ column; H₂O/CH₃CN gradient+0.1% trifluoroacetic acid;retention times: DNP-PA, 5.0 mins; DNP-PC, 4.26 mins). Conversion ofDNP-PA to DNP-PC is observed only in the presence of both YAD and NADP+.Reactions are also followed spectrophotometrically; the increase ofabsorbance at 340 nm indicated that NADP+ is simultaneously converted toNADPH.

The coupled DHFR-YAD reaction is followed using the same HPLC assay. Theinitial reaction mixture contained the substrates for DHFR-NADPH (50 μM)and 7-8-dihydrofolate (H₂F; 50 μM), YAD (Sigma, 0.5 unit) and DNP-PA (50μM) in buffer pH 7.7 (100 mM inidazole, 5 mM β-mercaptoethanol and 25 mMKCl). Conversion of DNP-PA to DNP-PC is observed when DHFR is added tothe above reaction mixture (DHFR 5 nM, 83%.; 1.25 nM, 14.5% after 32mins).

The concentration of DHFR obtained in the compartmentalised in vitrotranslation is in fact much higher than 5 nM (see Example 4). Theconversion of DNP-PA to DNP-PC is negligible in the absence of DHFR, orwhen methotrexate (MTX)—a potent inhibitor of the enzyme—is present (10μM). Hence, the formation of the secondary product, DNP-PC, is thereforelinked to the presence of the DHFR.

Using this coupled reaction, proteins conferring DHFR activity can beselected by: i) linking the genes to antibodies that specifically bindthe carboxylic product of DNP-PA, and ii) isolating these genes byaffinity purification using an anti-DNP antibody.

This approach is demonstrated by a routine immuno assay based on thecatELISA (Tawfik et al., 1993). Microtiter plates are coated withanti-rabbit immunoglobulins (Sigma, 10 μg/well) followed by rabbitpolyclonal serum that specifically bind glutaric acid derivatives(Tawfik et al., 1993) diluted 1:500 in phosphate saline buffer+1 mg/mlBSA). The plates are rinsed and blocked with BSA. The coupled reactionmixtures described above are diluted in Tris/BSA buffer (50 mM Tris, 150mM sodium chloride, 10 mg/ml BSA, pH 7.4) and incubated for 1 hr. Theplate is rinsed and an anti-DNP antibody (mouse monoclonal SPE21.11)diluted in the same buffer (1:10,000) is added and incubated for anhour. The plate is rinsed and peroxidase labelled anti mouse antibody(Jackson) is added followed by a peroxidase substrate (BM Blue;Boehringer Mannheim). A specific signal is observed only in the coupledreactions samples that contained DHFR (in addition to H₂F, NADPH, YADand DNP-PA).

Highly specific anti-carboxylic acid antibodies (Tawfik et al., 1993)are used for selection in two formats.

In the first, the anti-carboxylic acid antibody is coupled chemically toa high molecular weight avidin (or streptavidin) containing complex suchas that described in Example 5. Biotinylated DNA encoding DHFR iscoupled to this complex via the avidin-biotin interaction as describedin Example 5. This complex is then used in a compartmentalised coupledtranscription/translation system which also contains YAD and a taggedYAD substrate such as DNP-PA. If there is DHFR activity in thecompartment the DNP-PA is converted to DNP-PC. The anti-carboxylic acidantibodies, coupled to the DNA via the high molecular weight complexwill capture only DNP-PC molecules and not aldehyde molecules. DNA fromthose compartments containing active DHFR (and hence encoding activeDHFR if there is only one molecule of DNA per compartment) are thenaffinity purified by using anti-DNP antibodies.

In the second format, multiple streptavidin molecules are coupledtogether in a high molecular weight complex which can easily be coupledto biotinylated DNA encoding DHFR (see Example 5). This complex is usedin a compartmentalised coupled transcription/translation system whichalso contains YAD and a YAD substrate such asMeNPOC-biotin-benzaldehyde. The biotin group inMeNPOC-biotin-benzaldehyde is “caged” (Sundberg et al., 1995; Pirrungand Huang, 1996), that is, it cannot be bound by avidin or streptavidinuntil a photoremovable nitrobenzyl group has been cleaved off byirradiation with light. If there is DHFR activity in the compartment theMeNPOC-biotin-benzaldehyde is converted to MeNPOC-biotin-benzoic acid.After the compartmentalised reaction has run for a while the reaction isirradiated with light and the nitrobenzyl group removed and the compoundwill bind to the streptavidin-DNA complex. DNA in those compartmentscontaining active DHFR (and hence encoding active DHFR if there is onlyone molecule of DNA per compartment) is complexed with biotin-benzoicacid (instead of biotin-benzaldehyde) and can be affinity purified usingimmobilised anti-benzoic acid antibodies.

The presence of other enzymes which can catalyse the oxidation NAD+ orNADP+ to NADH or NADPH in the in vitro transcriptionl translation systemcan under certain circumstances make it difficult to use this YAD systemfor selection directly in the compartmentalised in vitrotranscription/translation system. In this case the selection is carriedout using the two-step compartmentalisation system described earlier.That is, the DHFR is first translated in compartments and then linked tothe DNA in the same compartment by means of a suitable affinity tag. Theemulsion is broken, the contents of the compartments pooled and the DNAaffinity purified away from the other components of thetranscription/translation system (including contaminatingoxido-reductases), by using antibodies specific to a digoxigenin ‘tag’attached to one end of the DNA molecule. The purified DNA molecules,together with the attached DHFR protein are then put into a reactionmixture contained the substrates for DHFR-NADPH (50 μM) and7-8-dihydrofolate (H₂F; 50 μM), YAD (Sigma, 0.5 unit) and DNP-PA (50 μM)in buffer pH 7.7 (100 mM imidazole, 5 mM β-mercaptoethanol and 25 mMKCl) and the reaction re-compartmentalised by emulsification to giveonly one, or at most a few, molecules of DNA per compartment.Anti-carboxylic acid antibodies (Tawfik et al., 1993) are used forselection in either of the two formats described above.

Example 9

Methylation of Genetic Elements by Gene Products

DNA methyltransferases, produced by in vitro transcription/translationin the aqueous compartments of a water-in-oil emulsion, methylate theDNA molecules which encode them in the compartments.

Selecting proteins with binding or catalytic activities using thecompartmentalisation system described here presents two basicrequirements: i) a single molecule of DNA (or at most a few molecules)encoding the proteins to be selected is expressed in a biologicallyactive form by a coupled transcription/translation system in the aqueouscompartments of a water-in-oil emulsion; and, ii) the protein to beselected must be able to modify the genetic element that encoded it insuch a way as to make it selectable in a subsequent step. In thisExample, we describe a group of proteins—DNA methyl transferases (typeII)—that are produce efficiently in the aqueous compartments of awater-in-oil emulsion system using a coupled transcriptionl/translationsystem. Furthermore, the in vitro translated DNA methyltransferasesefficiently modify the DNA molecules which encode them in situ in theaqueous compartments so that they can be selected and amplified. Thetarget sites on the DNA molecules are modified by methylation of acytosine at the C5 position which renders the sites resistant tocleavage by the cognate restriction endonuclease (i.e. HhaI for M.HhaI,and HaeIII for M.HaeIII). Hence, methylated DNA is selectable overnon-methylated DNA by virtue of its resistance to restrictionendonuclease cleavage.

The gene encoding M.HhaI is amplified by PCR using oligonucleotidesHhaI-Fo2S and HhaI-Bc directly from Haemophilius parahaemolyticus (ATCC10014). The gene encoding M.HaeIII is amplified by PCR usingoligonucleotides HaeIII-Fo2s and HaeII-Bc (SEQ. ID. No. 4) directly fromHaemophilus influenzae (biogroup aegyptius) (ATCC 11116). Both PCRfragments are cloned into the vector pGEM-4Z (Promega) digested withHindIII and KpnI downstream of the lac promoter and T7 RNA polymerasepromoter. The oligonucleotides HhaI-Bc and HaeIII-Bc (SEQ. ID. No. 4)append the efficient phage T7 gene 10 translational start site upstreamof the methyltransferase gene start codon. Oligonucleotide HhaI-Foappends an HhaI methylation/restriction site (M/R) and a HaeIII (INotI)site to function as substrates for M.HhaI and M.HaeIII respectively.Oligonucleotide HaeIII-Fo appends a NotI/HaeIII M/R site which functionsas a substrate for M.HaeIII (the M.HaeIII gene already contains twointernal HhaI M/R sites). DNA sequencing identifies clones with thecorrect nucleotide sequence.

The pGEM-M.HhaI and pGEM-M.HaeIII plasmids described above are amplifiedby PCR using primers LMB2-Biotin (SEQ. ID. No. 9) and LMB3-DIG (SEQ. ID.NO. 10) as above to create either 1167 base pair DIG-M.HhaI-Biotin or a1171 base pair DIG-M.HaeIII-Biotin DNA fragment, labelled at one end bybiotin and the other end by digoxigenin, and which carry the T7 RNApolymerase promoter, the phage T7 gene 10 translational start site, themethyltransferase gene and M/R sites of HaeIII and HhaI. The PCRfragments are each purified directly using Wizard PCR Preps (Promega).

The genes required for the coupled in vitro transcription-translation ofM.EcoRI and. M.EcoRV are amplified by PCR using plasmids pMB1 (Betlachet al., 1976) and pLB1 (Bougueleret et al., 1984) respectively, astemplates, a back primer appending the phage T7 gene 10 translationalstart site and LMB3 upstream of the methyltransferase gene ribosomebinding site (EcoRI-Bc or EcoRV-Bc) and a forward primer (EcoRI-Fo orEcoRI-Fo) appending LMB2. These fragments are further amplified by PCRusing primers LMB2-Biotin (SEQ. ID. No. 9) and LMB3-DIG (SEQ. ID. NO.10) as described above to create the DIG-M.Eco RI-Biotin andDIG-M.EcoRV-Biotin DNA fragments which carry the T7 RNA polymerasepromoter, the phage T7 gene 10 translational start site, themethyltransferase gene and M/R sites of EcoRI and EcoRV. These PCRfragments are each purified directly using Wizard PCR Preps (Promega).

The PCR-amplified DNA-methylases genes described above are expressed ina prokaryotic in vitro coupled transcription/translation system designedfor linear templates (Lesley et al., 1991). A commercial preparation ofthis system is used (E. coli S30 Extract System for Linear Templates;Promega) supplemented with T7 RNA polymerase and S-adenosyl methionine(SAM) at 80 μM concentration.

Methylation is assayed by measuring the resistance of DNA fragmentslabelled with DIG and biotin to cleavage by the cognate restrictionenzyme using the Boehringer-Mannheim DIG-Biotin ELISA or withradioactively labelled DNA fragments and streptavidin coated magneticbeads. In vitro reaction mixtures containing DIG-Biotin labelledfragments reacted in situ by coupled in vitro transcription-translationas described below are diluted in 1×W&B buffer (1M NaCl, 10 mM Tris, 1mM EDTA, pH 7.4)+0.1% Tween-20 (the concentration of the DIG/Biotinlabelled DNA in the assay is in the range of 0-250 pM) and incubated instreptavidin coated microtiter plates (high capacity) for 30-60 mins.The plate is rinsed (3 times 2×W&B and finally with 50 mM Tris pH 7.4+5mM MgCl₂) and the restriction enzymes (NEB) are added (10-50 unitsenzyme in 0.2 ml of the corresponding buffer) and incubated at 37° for3-12 hrs. The plate is rinsed and peroxidase-linked anti-DIG antibodies(diluted 1:1,500 in PBS+0.1% Tween-20+2 mg/ml BSA) are added for 40-60min followed by the peroxidase substrate (BM Blue; 70 μl/well). Theabsorbance (at 450 minus 650 nm) is measured after quenching with 0.5MH₂SO₄ (130 μl/well).

For the radioactive assay, the plasmids and PCR fragments describedabove are amplified by PCR using primers LMB2-Biotin (SEQ. ID. No. 9)and LMB3 and α-P³²-CTP to give P³²-labelled DNA fragments labelled atone end by biotin and which carry the T7 RNA polymerase promoter, thephage T7 gene 10 translational start site, the methyltransferase geneand the relevant M/R sites. These PCR fragments are purified directlyusing Wizard PCR Preps (Promega). Reaction mixtures containing theBiotin-P³²-labelled DNA reacted in situ by coupled in vitrotranscription-translation are diluted in 1×W&B buffer+0.1% Tween-20 andincubated with streptavidin coated magnetic beads (Dynal, M-280; 1-5×10⁶beads) for 30-60 mins. The beads are separated and rinsed (3 times2×W&B+0.1% Tween-20+3% BSA and finally with 50 mM Tris pH 7.4+5 mMMgCl₂). The restriction enzymes (NEB) are added (10-50 units enzyme in50-150 μl of the corresponding buffer) and incubated at 37° for 5-20hrs. The supernatant is removed and the beads rinsed and resuspended in100 μl water. The amount of radioactively-labelled DNA on the beads andin the supernatants is determined by scintillation.

All four methylases described here—M.HaeIII, M.HhaI, M.EcoRI andM.EcoRV—are expressed and active in the in vitro coupledtranscription/translation. Furthermore, the in vitro translatedmethylase can methylate its own gene thus rendering it resistant tocleavage by the cognate methylase (self-methylation). Both processes,the coupled in vitro transcription-translation of the methylase gene aswell as its methylation proceed efficiently in the same reactionmixture. More specifically, DNA fragments (at 0.5 to 10 nMconcentrations) which carry the T7 RNA polymerase promoter, the phage T7gene 10 translational start site, a methyltransferase gene and M/R sitesof all four methylases become resistant to cleavage by the cognaterestriction endonuclease. For example, the DNA fragment encoding M.EcoRImethyltransferase becomes resistant to cleavage by EcoRI (75-100% after20-90 minutes at 25° C.) when incubated with E. coli S30 Extract Systemfor Linear Templates (Promega), SAM (80 μM) and T7 RNA polymerase. Theresistance to cleavage as a result of methylation is selective andspecific: under the same conditions, resistance to cleavage by HhaI orM.EcoRV is not observed; moreover, resistance to cleavage by EcoRI isnot observed when translation is inhibited (e.g. in the presence ofpuromycin or in the absence of T7 RNA polymerase). Similar results whereobtained when survival of the genes is assayed by DIG-Biotin ELISA orwith Biotin-P³²-labelled DNA fragments as described above. Methylationin trans, i.e., of DNA fragments (other than those encoding for thecognate methylase) appending M/R sites is also observed in the E. coliS30 coupled in vitro transcription-translation system in the presence ofa gene encoding for a methylase.

Both processes, the coupled in vitro transcription-translation of themethylase genes as well as their self-methylation proceed efficiently inthe aqueous compartments of a water-in-oil emulsion. More specifically,DNA fragments (at 0.1-10 nM concentrations) which carry the T7 RNApolymerase promoter, the phage T7 gene 10 translational start site, themethyltransferase gene (for example, M.HhaI) and the M/R sites ofHaeIII, HhaI and EcoRI are added to E. coli S30 Extract System forLinear Templates (Promega) in the presence of SAM (80 μM) and T7 RNApolymerase. The ice cooled reaction mixtures are emulsified byhomogenising for 1 minute with an Ultra-Turrax T25 disperser at 20,000rpm as described in Example 1 and incubated at 25°-30° for 0-180 mins.The reaction is stopped and the aqueous phase is separated (seeExample 1) and the methylation of the DIG-Biotin or Biotin-P³²-labelledDNA fragments is assayed as described above. Methylation of up to 20% ofthe compartmentalised genes to cleavage by HhaI is observed after 60-180mins incubation. No resistance is observed when the ice-cold emulsion isbroken just after it is made and the reaction quenched by etherextraction (‘0 mins’). The methylation is selective: under the sameconditions, resistance to cleavage by HaeIII or EcoRI is not observed.Moreover, the assay of P³²-labelled DNA fragments has shown thatself-methylation of both M.HaeIII and M.HhaI proceed at concentrationsof genes that correspond to an average of less than one gene percompartment (0.1-0.5 nM; see Example 4). Thus, the coupled in vitrotranscription-translation of the methylases genes as well as theirself-methylation proceed efficiently even when only a single geneticelement is present in aqueous compartments of the water-in-oil emulsion.

HaeIII methylase activity resulting from coupledtranscription/translation of M.HaeIII genes is also measured in thelarger microcapsules produced by stirring alone, and by stirringfollowed by further homogenisation with an Ultra-Turrax T25 disperser at8,000 rpm, 9,000 rpm, or 13,500 rpm for 1 minute as described inExample 1. The results are presented in FIG. 2 b. The concentration ofM.HaeIII genes used (2.5 nM) gives an average of 1, 1.5 and 4.8 geneticelements per droplet in the emulsions homogenised at 13,500 rpm, 9,500rpm and 8,000 rpm, respectively, and an average of 14 genetic elementsper droplet in the emulsion prepared by stirring only. The addition ofan anionic surfactant—e.g., sodium deoxycholate, typically to 0.5%(w/v), to the in vitro translation mixture significantly increases thepercentage of genetic elements methylated in the emulsions.

Example 10

Genetic Elements Encoding DNA Methyltransferases can be Selected andAmplified Following Their Self-Methylation in the Aqueous Compartmentsof a Water-in-oil Emulsion.

The methylation of genes encoding for DNA-methylases allows them to beisolated and amplified in a subsequent step. The methylated DNA isselectable over non-methylated DNA by virtue of its resistance torestriction endonuclease cleavage. Thus, the genetic elements thatremain intact after treatment with the cognate restriction enzyme can beamplified by PCR. However, such a selection is obviously unattainable ifother genes that contain the same R/M site but do not encode for themethylase are present in same reaction mixture. This is becausecross-methylation of the non-methylase genes (that are present at alarge excess) will render them resistant to cleavage by the cognaterestriction enzyme and thus amplifiable by PCR. Under these conditions,selection of genes encoding the. methylase will become possible only ifthey are compartmentalised—namely, if only one, or few genes are presentin a single compartment so that self methylation is the major process inthat compartment. Cross-methylation is avoided since non-methylase genesthat are present in compartments that do not contain a methylase genewill remain un-methylated.

The genes used in the experiment are a 1194 base pair M.HaeIII fragment(DIG-M.HaeIII-3s-Biotin) encoding methylase HaeIII and a 681 base pairfolA fragment (DIG-folA-3s-Biotin) encoding the enzyme dihydrofolatereductase (DHFR) containing additional HaeIII and HhaIrestriction/modification sites (See FIG. 1 b). Both DNA fragments arelabelled at one end with digoxigenin (DIG) and the other with biotin,and contain a T7 RNA polymerase promoter (T7 Promoter) and T7 gene 10translational initiation site (rbs) for expression in vitro.

pGEM-4Z-3s is created by annealing oligonucleotides HaeHha-P1 andHaeHha-Mi (SEQ. ID. No. 2) (Table 1) and ligating them into HindIII andEcoRI cut pGEM-4Z (Promega). The M.HaeIII gene is amplified by PCR fromHaemophilus influenzae (biogroup aegyptius) (ATCC 11116) usingoligonucleotides HaeIII-FoNC (SEQ. ID. No. 3) and HaeIII-Bc (SEQ. ID.No. 4) (Table 1). The folA gene is amplified from Escherichia coli usingprimers EDHFR-Fo (SEQ. ID. No. 5) and EDHFR-Ba (SEQ. ID. No. 6) (Table1). Both amplified genes are digested with HindIII and KpnI and clonedinto pGEM-4Z-3s, creating the expression vectors pGEM-HaeIII-3s andpGEM-folA-3s. DIG-M.HaeIII-3s-Biotin and DIG-folA-3s-Biotin (see FIG. 1b) are amplified from these vectors by PCR with pfu polymerase usingprimers LMB2-Biotin (SEQ. ID. No. 9) and LMB3-DIG (SEQ. ID. NO. 10) (20cycles) and purified using Wizard PCR Preps (Promega). DIG-D1.3-Biotin,a 942 bp DNA fragment containing four HaeIII R/M sites used as asubstrate to assay HaeIII methylase activity, is amplified from a pUC19derivative containing a D1.3 single-chain Fv gene (McCafferty et al.,1990) as above. A 558 bp carrier DNA (g3 carrier DNA; an internalfragment of phage fd gene III which has no T7 promoter, HaeIII or HhaIR/M sites) is amplified by PCR with Taq polymerase from pHEN1 DNA(Hoogenboom et al., 1991) using primers G3FRAG-Fo (SEQ. ID. No. 11) andG3FRAG-Ba (SEQ. ID. No. 12) (Table 1) and purified by phenol-chloroformextraction and ethanol precipitation. This DNA (at≧10 nM) was used as acarrier in dilution of all DNA used for the reactions in this example.

FIG. 3 demonstrates the selection of M.HaeIII genes encoding the DNAmethylase HaeIII from an excess of folA genes (encoding DHFR which doesnot methylate DNA). Both genes have the same HaeIII R/M sequencesappended to act as a substrate (FIG. 1 b). After translation in theaqueous compartments of an emulsion the HaeIII R/M sequences attached tomethylase genes are methylated. These genes are rendered resistant tocleavage by HaeIII endonuclease and are subsequently amplified by PCR.folA genes, present in other compartments, remain unmethylated, arecleaved and not amplified. The PCR products are analysed by agarose gelelectrophoresis where enrichment for the M.HaeIII genes can bevisualised by the appearance of a 1194 bp band (FIG. 3).

The E. coli S30 extract system for linear DNA (Promega) is used,supplemented with g3 carrier DNA (10 nM), DNA fragments(DIG-M.HaeIII-3s-Biotin and DIG-M.folA-3s-Biotin at ratios andconcentrations indicated below), T7 RNA polymerase (10⁴ units), sodiumdeoxycholate (Fluka, 0.5% w/v; in emulsified reactions only) andS-adenosyl methionine (NEB, 80 μM). Reactions are set up using DNAfragments DIG-M.HaeIII-3s-Biotin and DIG-M.folA-3s-Biotin at a ratio of1:10³ and at a total concentration of 200 pM (FIG. 3A) and ratios of1:10⁴ to 1:10⁷ and a total concentration of 500 pM as described inExample 1. Reactions are incubated for 2 hours at 25° C. To recover thereaction mixtures, the emulsions are spun at 3,000 g for 5 minutes andthe oil phase removed leaving the concentrated emulsion at the bottom ofthe vial. Quenching buffer (0.2 ml of 25 μg/ml yeast RNA in W+B buffer:1 M NaCl, 10 mM Tris-HCl, 1 mM EDTA pH 7.4) and 2 ml of water-saturatedether are added and the mixture is vortexed, centrifuged briefly, andthe ether phase removed. The aqueous phase is washed with ether anddried (5 minutes in a SPEEDVAC™ evaporator centrifuge (THERMOSAVANT) atambient temperature). DNA is captured on 100 μg M-280 streptavidinDYNABEADS™ (superparamagnetic particles; DYNAL; 2 hours at ambienttemperature). The DYNABEADS™ are washed sequentially with: W+B buffer;2.25 M Guanidine-HCl, 25 mM Tris-HCl, pH 7.4; W+B buffer; and, twicewith restriction buffer. Beads are resuspended in 100 μl restrictionbuffer containing 10 units HaeIII (or HhaI) and incubated at 37° C. for5 hours. The beads are washed three times with W+B buffer, twice with 50mM KCl, 10 mM Tris-HCl, 0.1% Triton X-100, pH 9.0, and then resuspendedin 100 μl of the same buffer supplemented with 1.5 mM MgCl₂ (PCRbuffer). Aliquots of beads (2-20 μl) are amplified by PCR using Taqpolymerase added at 94° C. with primers LMB2-Biotin and LMB3-DIG (50 μlreactions; 32 cycles of 1 minute at 94°, 1 minute at 55°, 2 minutes at72°). This DNA is purified using WIZARD™ PCR Preps (PCR purificationkit; PROMEGA) and used for the second round of selection (20 pM in the1:10⁴ and 1:10⁵ selections and 500 pM in the 1:10⁶ and 1:10⁷selections). For gel electrophoresis and activity assays this DNA(diluted to˜1 pM) is further amplified with primers LMB2-Nest andLMB3-Nest which anneal immediately inside LMB2 and LMB3 respectively (25cycles of 1 minute at 94°, 1 minute at 50°, 1.5 minutes at 72°) andpurified as above. This DNA (at 10 nM), which has neither DIG nor Biotinappended, is also translated in vitro in the presence of 10 nMDIG-D1.3-Biotin, a 942 bp DNA containing four HaeIII R/M sites.Methylation of the DIG-D1.3-Biotin substrate is determined by DIG-BiotinELISA as Example 9.

A single round of selection of a 1:1000 ratio of M.HaeIII:folA genes inthe emulsion results in a roughly 1:1 final gene ratio (FIG. 3 a).Several control experiments indicate that selection proceeds accordingto the mechanism described above: a band corresponding to the M.HaeIIIgene is not observed when the initial mixture of genes is amplified byPCR; nor after reaction in solution (non-emulsified); nor whenemulsified in the absence of transcription/translation (when T7 RNApolymerase is onitted); nor when the reacted genes are cleaved at R/Msites other than those of HaeIII—e.g., after digestion with HhaI. Theyield of M.HaeIII DNA after selection is less than 100% primarily due toincomplete digestion by HaeIII rather than cross-methylation asindicated by the large folA band observed in the absence of methylaseactivity (when T7 polymerase is not added). During digestion, theconcentration of DNA drops well below the K_(M) of HaeIII (6 nM) anddigestion becomes extremely inefficient.

A band corresponding to M.HaeIII genes also becomes visible after asingle-round of selection starting from M.HaeIII:folA ratios of 1:10⁴ to1:10⁵ (FIG. 3 b), but not at lower ratios, indicating an enrichmentfactor of at least 5000-fold. Selection of a small number of genes froma large pool (e.g., a gene library) therefore requires further rounds ofselection. When the HaeIII-digested and amplified DNA from the firstround of selection is subjected to a second round of selection, a bandcorresponding to M.HaeIII genes also became visible from 1:10⁶ and 1:10⁷starting ratios of M.HaeIII:folA. A second round of selection is alsoperformed on the DNA derived from the 1:10⁴ to 1:10⁵ starting ratios ofM.HaeIII:folA. This gives a further enrichment, up to a ratio ofapproximately 3:1 in favour of the M.HaeIII genes. Before and after eachround of selection the genes are amplified, translated in vitro andreacted with a separate DNA substrate to assay for HaeIII methylaseactivity. These assays indicate that enrichment for the M.HaeIII genesas observed by gel electrophoresis results in a parallel increase inHaeIII methylase activity (FIG. 3 b).

TABLE 1 Oligonucleotides HaeHha-Pl 5′-AGC TTG CAT GCC TGC GGT ACC GGCCAT GCG CAT GGC CTA GCG CAT GCG GCC GCT AGC GCG-3′ (SEQ. ID. No. 1)HaeHha-Mi 5′-AAT TCG CGC TAG CGG CCG CAT GCG CTA GGC CAT GCG CAT GGC CGGTAC CGC AGG CAT GCA-3′ (SEQ. ID. No. 2) HaeIII-FoNC 5′-CGA GCT AGA GGTACC TTA TTA ATT ACC TTT ACA AAT TTC CAA TGC AGA TTT TAT-3′ (SEQ. ID. No.3) HaeIII-Bc 5′-GCA TCT GAC AAG CTT AAT AAT TTT GTT TAA CTT TAA GAA GGAGAT ATA CAT ATG AAT TTA ATT AGT CTT TTT TCA GGT GCA GGG-3′ (SEQ. ID. No.4) EDHFR-Fo 5′-CGA GCT AGA GGT ACC TTA TTA CCG CCG CTC CAG AAT CTC AAAGCA ATA G-3′ (SEQ. ID. No. 5) EDHFR-Ba 5′-GCA TCT GAC AAG CTT AAT AATTTT GTT TAA CTT TAA GAA GGA GAT ATA CAT ATG ATC AGT CTG ATT GCG GCG TTAGCG GTA G-3′ (SEQ. ID. No. 6) LMB2-Nest 5′-GAA TTG GAT TTA GGT GAC-3′(SEQ. ID. No. 7) LMB3-Nest 5′-CAT GAT TAC GCC AAG CTC-3′ (SEQ. ID. No.8) LMB2-Biotin 5′-Biotin-GTA AAA CGA CGG CCA GT-3′ (SEQ. ID. No. 9)LMB3-DIG 5′-Digoxigenin-CAG GAA ACA GCT ATG AC-3′ (SEQ. ID. No. 10)G3FRAG-Fo 5′-GTC TCT GAA TTT ACC GTT CCA G-3′ (SEQ. ID. No. 11)G3FRAG-Ba 5′-GAA ACT GTT GAA AGT TGT TTA G-3′ (SEQ. ID. No. 12) P2T7Ba5′-ATT ATA ATA CGA CTC ACT ATA GGG AGA GTT ATC AGG CAT GCA CC-3′ (SEQ.ID. No. 13) P9Fo 5′-CTA GCT CCC ATT AAG GAG-3′ (SEQ. ID. No. 14) LMB25′-GTA AAA CGA CGG CCA GT-3′ (SEQ. ID. No. 15) LMB3 5′-CAG GAA ACA GCTATG AC-3′ (SEQ. ID. No. 16) HhaI-Fo2S 5′-CGA GCT AGA GGT ACC GCG GCC GCTGCG CTT ATT AAT ATG GTT TGA AAT TTA ATG ATG AAC CAA TG-3′ (SEQ. ID. No.17) HhaI-Bc 5′-GCA TCT GAC AAG CTT AAT AAT TTT GTT TAA CTT TAA GAA GGAGAT ATA CAT ATG ATT GAA ATA AAA GAT AAA CAG CTC ACA GG-3′ (SEQ. ID. No.18) HaeIII-Fo2s 5′-CGA GCT AGA GGT ACC GCG GCC GCT GCG CTT ATT AAT TACCTT TAC AAA TTT CCA ATG CAG ATT TTA T-3′ (SEQ. ID. No. 19) EcoRI-Bc5′-CAG GAA ACA GCT ATG ACA AGC TTA ATA CGA CTC ACT ATA GGG AGA TAT TTTTTA TTT TAA TAA GGT TTT AAT TAA TGG-3′ (SEQ. ID. No. 20) EcoRI-Fo 5′-GTAAAA CGA CGG CCA GTG AAT TCT TAT TAC TTT TGT AAT CGT TTG TTT TTT ATC-3′(SEQ. ID. No. 21) EcoRV-Bc 5′-CAG GAA ACA GCT ATG ACA AGC TTA ATA CGACTC ACT ATA GGG AGA AAT GGG TTT CTT TGG CAT ATT TTT TAC AAA TG-3′ (SEQ.ID. No. 22) EcoRV-Fo 5′-GTA AAA CGA CGG CCA GTG AAT TCG ATA TCT TAT TACTCT TCA ATT ACC AAA ATA TCC CC-3′ (SEQ. ID. No. 23) LMB2-Biotin has a5′-terminal biotin linked by a 16-atom spacer arm. LMB3-DIG has a5′-terminal digoxygenin linked by a 12-atom space arm. Oligonucleotideslabelled at the 5′ terminus with Biotin or Digoxigenin were purchasedfrom Eurogentec.

RERERENCES

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1. A method for isolating one or more genetic elements encoding a geneproduct having a desired activity, comprising the steps of: (a)expressing the genetic elements to produce their respective geneproducts such that the gene products are linked to the genes encodingthem; (b) compartmentalizing the genetic elements into microcapsules;(c) sorting the genetic elements which produce gene product(s) havingthe desired activity.
 2. The method according to claim 1 wherein a geneproduct with the desired activity induces a change in the microcapsulewhich enables the microcapsule containing the gene product and thegenetic element encoding it to be sorted.
 3. The method according toclaim 2 wherein a gene product having the desired activity modifies oneor more molecules within the microcapsule which enables the microcapsulecontaining the gene product and the genetic element encoding it to besorted.
 4. The method according to claim 2 wherein the gene producthaving the desired activity induces a change in the optical propertiesof the microcapsule and the microcapsules are sorted thereby.
 5. Themethod according to claim 4 wherein the change in optical properties isa change in fluorescence.
 6. The method according to claim 1, whereinthe desired activity of the gene product within the microcapsuleresults, directly or indirectly, in the modification of the geneticelement encoding the gene product to enable the isolation of the geneticelement.
 7. The method according to claim 6 wherein the gene producthaving the desired activity induces a change in the microcapsule which,when detected, triggers the modification of the genetic element withinthe compartment to enable the isolation of the genetic element.
 8. Themethod according to claim 7 wherein the change in the microcapsule is achange in the optical properties thereof.
 9. The method according toclaim 8, wherein the change in optical properties is a change influorescence.
 10. The method according to claim 6 wherein a part of thegenetic element is a substrate and the activity of the desired geneproduct within the microcapsule results, directly or indirectly, in theconversion of said substrate into a product which remains part of thegenetic element and enables its isolation.
 11. The method according toclaim 6 wherein the activity of the desired gene product within themicrocapsule results, directly or indirectly, in the conversion of asubstrate into a product, and the genetic element further comprises aligand which binds to said product and enables its isolation.
 12. Themethod according to claim 6 wherein the genetic element comprises aligand and the desired gene product within the microcapsule binds tosaid ligand to enable the isolation of the genetic element.
 13. Themethod according to claim 1 wherein the activity of the gene productwithin the microcapsule results in the alteration of the expression of asecond gene within the compartment and the activity of the product ofthe said second gene enables the isolation of the genetic element. 14.The method according to claim 1, wherein the genetic elements areisolated from a library of genetic elements encoding a repertoire ofgene products.
 15. The method according to claim 1 further comprisingthe additional step of: (d) introducing one or more mutations into thegenetic element(s) isolated in step (c).
 16. The method according toclaim 15 further comprising iteratively repeating one or more of steps(a) to (d).
 17. The method according to claim 1 further comprisingamplifying the genetic elements.
 18. The method according to claim 1,wherein compartmentalizing the genetic elements into microcapsules isachieved by forming a water-in-oil emulsion of an aqueous solution in anoil-based medium.
 19. The method according to claim 1, wherein thegenetic elements are sorted by affinity purification.
 20. The methodaccording to claim 1, wherein the genetic elements are sorted byselective ablation of genetic elements which do not encode the geneproduct having the desired activity.
 21. A method for preparing a geneproduct, comprising the steps of: (a) preparing a genetic clementencoding the gene product; (b) expressing the genetic elements toproduce their respective gene products such that the gene products arelinked to the genetic elements encoding them; (c) compartmentalizinggenetic elements into microcapsules; (d) sorting the genetic elementswhich produce the gene product(s) having the desired activity; and (e)expressing the gene product having the desired activity.